Methods, kits and compositions pertaining to linear beacons

ABSTRACT

This invention is directed to methods for determining amplified nucleic acid using Linear Beacons.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/610,337,filed Jun. 30, 2003, now U.S. Pat. No. 7,422,852, which is a divisionalof application Ser. No. 09/950,459, filed Sep. 10, 2001, now U.S. Pat.No. 6,649,349, which is a divisional of application Ser. No. 09/179,162,filed Oct. 26, 1998, now U.S. Pat. No. 6,485,901, which claims thebenefit of U.S. Provisional Application No. 60/063,283, filed Oct. 27,1997, which are incorporated herein by reference in their entirety forall purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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BACKGROUND OF THE INVENTION

This invention is related to the field of probe-based nucleic acidsequence detection, analysis and quantitation. More specifically, thisinvention relates to novel methods, kits and compositions pertaining toLinear Beacons.

Quenching of fluorescence signal can occur by either FluorescenceResonance Energy Transfer “FRET” (also known as non-radiative energytransfer: See: Yaron et al., Analytical Biochemistry 95: 228-235 (1979)at p. 232, col. 1, lns. 32-39) or by non-FRET interactions (also knownas radiationless energy transfer; See: Yaron et al., AnalyticalBiochemistry 95 at p. 229, col. 2, lns. 7-13). The criticaldistinguishing factor between FRET and non-FRET quenching is thatnon-FRET quenching requires short range interaction by “collision” or“contact” and therefore requires no spectral overlap between themoieties of the donor and acceptor pair (see: Yaron et al., AnalyticalBiochemistry 95 at p. 229, col. 1, lns. 22-42). Conversely, FRETquenching requires spectral overlap between the donor and acceptormoieties and the efficiency of quenching is directly proportional to thedistance between the donor and acceptor moieties of the FRET pair (see:Yaron et al., Analytical Biochemistry 95 at p. 232, col. 1, ln. 46 tocol. 2, ln. 29). Extensive reviews of the FRET phenomenon are describedin Clegg, R. M., Methods Enzymol., 221: 353-388 (1992) and Selvin, P.R., Methods Enzymol., 246: 300-334 (1995). Yaron et al. also suggestedthat the principles described therein might be applied to the hydrolysisof oligonucleotides (see: Yaron et al., Analytical Biochemistry 95 at p.234, col. 2, lns. 14-18).

The FRET phenomenon has been utilized for the direct detection ofnucleic acid target sequences without the requirement that labelednucleic acid hybridization probes or primers be separated from thehybridization complex prior to detection (see: Livak et al. U.S. Pat.No. 5,538,848). One method utilizing FRET to analyze Polymerase ChainReaction (PCR) amplified nucleic acid in a closed tube format iscommercially available from Perkin Elmer. The TaqMan™ assay utilizes anucleic acid hybridization probe which is labeled with a fluorescentreporter and a quencher moiety in a configuration which results inquenching of fluorescence in the intact probe. During the PCRamplification, the probe sequence specifically hybridizes to theamplified nucleic acid. When hybridized, the exonuclease activity of theTaq polymerase degrades the probe thereby eliminating the intramolecularquenching maintained by the intact probe. Because the probe is designedto hybridize specifically to the amplified nucleic acid, the increase influorescence intensity of the sample, caused by enzymatic degradation ofthe probe, can be correlated with the activity of the amplificationprocess.

Nonetheless, this method preferably requires that each of thefluorophore and quencher moieties be located on the 3′ and 5′ termini ofthe probe so that the optimal signal to noise ratio is achieved (see:Nazarenko et al, Nucl. Acids Res. 25: 2516-2521 (1997) at p. 2516, col.2, lns. 27-35). However, this orientation necessarily results in lessthan optimal fluorescence quenching because the fluorophore and quenchermoieties are separated in space and the transfer of energy is mostefficient when they are close. Consequently, the background emissionfrom unhybridized probe can be quite high in the TaqMan™ assay (see:Nazarenko et al., Nucl. Acids Res. 25: at p. 2516, col. 2, lns. 36-40).

The nucleic acid Molecular Beacon is another construct which utilizesthe FRET phenomenon to detect target nucleic acid sequences (see: Tyagiet al. Nature Biotechnology, 14: 303-308 (1996). A nucleic acidMolecular Beacon comprises a probing sequence embedded within twocomplementary arm sequences (see: Tyagi et al, Nature Biotechnology, 14:at p. 303, col. 1, lns. 22-30). To each termini of the probing sequenceis attached one of either a fluorophore or quencher moiety. In theabsence of the nucleic acid target, the arm sequences anneal to eachother to thereby form a loop and hairpin stem structure which brings thefluorophore and quencher together (see: Tyagi et al., NatureBiotechnology, 14: at p. 304, col. 2, lns. 14-25). When contacted withtarget nucleic acid, the complementary probing sequence and targetsequence will hybridize. Because the hairpin stem cannot coexist withthe rigid double helix that is formed upon hybridization, the resultingconformational change forces the arm sequences apart and causes thefluorophore and quencher to be separated (see: Tyagi et al. NatureBiotechnology, 14: at p. 303, col. 2, lns. 1-17). When the fluorophoreand quencher are separated, energy of the donor fluorophore does nottransfer to the acceptor moiety and the fluorescent signal is thendetectable. Since unhybridized “Molecular Beacons” are non-fluorescent,it is not necessary that any excess probe be removed from an assay.Consequently, Tyagi et al. state that Molecular Beacons can be used forthe detection of target nucleic acids in a homogeneous assay and inliving cells (see: Tyagi et al., Nature Biotechnology, 14: at p. 303,col. 2; lns. 15-77).

The arm sequences of the disclosed nucleic acid Molecular Beaconconstructs are unrelated to the probing sequence (see: Tyagi et al.,Nature Biotechnology, 14: at p. 303, col. 1; ln. 30). Because the Tyagiet al. Molecular Beacons comprise nucleic acid molecules, proper stemformation and stability is dependent upon the length of the stem, theG:C content of the arm sequences, the concentration of salt in which itis dissolved and the presence or absence of magnesium in which the probeis dissolved (see: Tyagi et al., Nature Biotechnology, 14: at p. 305,col. 1; lns. 1-16). Furthermore, the Tyagi et al. nucleic acid MolecularBeacons are susceptible to degradation by endonucleases andexonucleases.

Upon probe degradation, background fluorescent signal will increasesince the donor and acceptor moieties are no longer held in closeproximity. Therefore, assays utilizing enzymes known to have nucleaseactivity, will exhibit a continuous increase in background fluorescenceas the nucleic acid Molecular Beacon is degraded (see: FIG. 7 in Tyagiet al: the data associated with (∘) and (□) demonstrates that thefluorescent background, presumably caused by probe degradation,increases with each amplification cycle.) Additionally, MolecularBeacons will also, at least partially, be degraded in living cellsbecause cells contain active nuclease activity.

The constructs described by Tyagi et al. are more broadly described inWO95/13399 (hereinafter referred to as “Tyagi2 et al.” except thatTyagi2 et al. also discloses that the nucleic acid Molecular Beacon mayalso be bimolecular wherein they define bimolecular as being unitaryprobes of the invention comprising two molecules (e.g.,oligonucleotides) wherein half, or roughly half, of the targetcomplement sequence, one member of the affinity pair and one member ofthe label pair are present in each molecule (see: Tyagi2 et al., p. 8,ln. 25 to p. 9, ln. 3). However, Tyagi2 et al. specifically states thatin designing a unitary probe for use in a PCR reaction, one wouldnaturally choose a target complement sequence that is not complementaryto one of the PCR primers (see: Tyagi2 et al., p. 41, ln. 27). Assays ofthe invention include real-time and end point detection of specificsingle-stranded or double stranded products of nucleic acid synthesisreactions, provided however that if unitary probes will be subjected tomelting or other denaturation, the probes must be unimolecular (see:Tyagi2 et al., p. 37, lns. 1-9). Furthermore, Tyagi2 et al. stipulatethat although the unitary probes of the invention may be used withamplification or other nucleic acid synthesis reactions, bimolecularprobes (as defined in Tyagi2 et al.) are not suitable for use in anyreaction (e.g., PCR) wherein the affinity pair would be separated in atarget-independent manner (see: Tyagi2 et al., p. 13, lns. 9-12).Neither Tyagi et al. nor Tyagi2 et al. disclose, suggest or teachanything about PNA.

In a more recent disclosure, modified hairpin constructs which aresimilar to the Tyagi et al. nucleic acid Molecular Beacons, but whichare suitable as primers for polymerase extension, have been disclosed(see: Nazarenko et al., Nucleic Acids Res. 25: 2516-2521 (1997)). Amethod suitable for the direct detection of PCR-amplified DNA in aclosed system is also disclosed. According to the method, the Nazarenkoet al. primer constructs are, by operation of the PCR process,incorporated into the amplification product. Incorporation into a PCRamplified product results in a change in configuration which separatesthe donor and acceptor moieties. Consequently, increases in theintensity of the fluorescent signal in the assay can be directlycorrelated with the amount of primer incorporated into the PCR amplifiedproduct. The authors conclude, this method is particularly well suitedto the analysis of PCR amplified nucleic acid in a closed tube format.

Because they are nucleic acids, the Nazarenko et al. primer constructsare admittedly subject to nuclease digestion thereby causing an increasein background signal during the PCR process (see: Nazarenko et al.,Nucleic Acids Res. 25: at p. 2519, col. 1; lns. 28-39). An additionaldisadvantage of this method is that the Molecular Beacon like primerconstructs must be linearized during amplification (see: Nazarenko etal., Nucleic Acids Res. 25: at p. 2519, col. 1, lns. 7-8). Consequently,the polymerase must read through and dissociate the stem of the hairpinmodified Molecular Beacon like primer construct if fluorescent signal isto be generated. Nazarenko et al. does not suggest, teach or discloseanything about PNA.

In still another application of FRET to target nucleic acid sequencedetection, doubly labeled fluorescent oligonucleotide probes which havebeen rendered impervious to exonuclease digestion have also been used todetect target nucleic acid sequences in PCR reactions and in-situ PCR(see: Mayrand, U.S. Pat. No. 5,691,146). The oligonucleotide probes ofMayrand comprise a fluorescer (reporter) molecule attached to a firstend of the oligonucleotide and a quencher molecule attached to theopposite end of the oligonucleotide (see: Mayrand, Abstract). Mayrandsuggests that the prior art teaches that the distance between thefluorophore and quencher is an important feature which must be minimizedand consequently the preferred spacing between the reporter and quenchermoieties of a DNA probe should be 6-16 nucleotides (see: col. 7, lns.8-24). Mayrand, however teaches that the reporter molecule and quenchermoieties are preferably located at a distance of 18 nucleotides (see:col. 3, Ins 35-36) or 20 bases (see: col. 7, lns. 25-46) to achieve theoptimal signal to noise ratio. Consequently, both Mayrand and the priorart cited therein teach that the detectable properties of nucleic acidprobes (DNA or RNA) comprising a fluorophore and quencher exhibit astrong dependence on probe length.

Resistance to nuclease digestion is also an important aspect of theinvention (see: U.S. Pat. No. 5,691,146 at col. 6, lns. 42-64) andtherefore, Mayrand suggests that the 5′ end of the oligonucleotide maybe rendered impervious to nuclease digestion by including one or moremodified internucleotide linkages onto the 5′ end of the oligonucleotideprobe (see: U.S. Pat. No. 5,691,146 at col. 6, lns. 45-50). Furthermore,Mayrand suggests that a polyamide nucleic acid (PNA) or peptide can beused as a nuclease resistant linkage to thereby modify the 5′ end of theoligonucleotide probe of the invention and render it impervious tonuclease digestion (see: U.S. Pat. No. 5,691,146 at col. 6, lns. 53-64).Mayrand does not however, disclose, suggest or teach that a PNA probeconstruct might be a suitable substitute for the practice of theinvention despite having obvious knowledge of its existence.Furthermore, Mayrand does not teach one of skill in the art how toprepare and/or label a PNA with the fluorescer or quencher moieties.

The efficiency of energy transfer between donor and acceptor moieties asthey can be influenced by oligonucleotide length (distance) has beenfurther examined and particularly applied to fluorescent nucleic acidsequencing applications (see: Mathies et al., U.S. Pat. No. 5,707,804).Mathies et al. states that two fluorophores will be joined by a backboneor chain where the distance between the two fluorophores may be varied(see: U.S. Pat. No. 5,707,804 at col. 4, lns. 1-3). Thus, the distancemust be chosen to provide energy transfer from the donor to the acceptorthrough the well-known Foerster mechanism (see: U.S. Pat. No. 5,707,804at col. 4, lns. 7-9). Preferably about 3-10 nucleosides separate thefluorophores of a single stranded nucleic acid (see: U.S. Pat. No.5,707,804 at col. 7, lns. 21-25). Mathies et al. does not suggest, teachor disclose anything about PNA.

From the analysis of DNA duplexes is has been observed that: 1: theefficiency of FET (or FRET as defined herein) appears to depend somehowon the nucleobase sequence of the oligonucleotide; 2: donor fluorescencechanges in a manner which suggests that dye-DNA interactions affect theefficiency of FET; and 3: the Foerster equation does not quantitativelyaccount for observed energy transfer and therefore the length betweendonor and acceptor moieties attached to oligonucleotides cannot bequantitated, though it can be used qualitatively (see: Promisel et al.,Biochemistry, 29: 9261-9268 (1990). Promisel et al. suggest thatnon-Foerster effects may account for some of their observed butotherwise unexplainable results (see: Promisel et al., Biochemistry, 29:at p. 9267, col. 1, ln. 43 to p. 9268, col. 1, ln. 13). The results ofPromisel et al. suggest that the FRET phenomena when utilized in nucleicacids in not entirely predictable or well understood. Promisel et al.does not suggest, teach or disclose anything about PNA and, in fact, themanuscript predates the invention of PNA.

The background art thus far discussed does not disclose, suggest orteach anything about PNA oligomers to which are directly attached a pairof donor and acceptor moieties. In fact, the FRET phenomenon as appliedto the detection of nucleic acids, appears to be confined to thepreparation of constructs in which the portion of the probe which iscomplementary to the target nucleic acid sequence is itself comprisedsolely of nucleic acid.

FRET has also been utilized within the field of peptides. (see: Yaron etal. Analytical Biochemistry 95 at p. 232, col. 2, ln. 30 to p. 234, col.1, ln. 30). Indeed, the use of suitably labeled peptides as enzymesubstrates appears to be the primary utility for peptides which arelabeled with donor and acceptor pairs (see: Zimmerman et al., AnalyticalBiochemistry, 70: 258-262 (1976), Carmel et al., Eur. J. Biochem., 73:617-625 (1977), Ng et al., Analytical Biochemistry, 183: 50-56 (1989),Wang et al., Tett. Lett., 31: 6493-6496 (1990) and Meldal et al.,Analytical Biochemistry, 195: 141-147 (1991). Early work suggested thatquenching efficiency of the donor and acceptor pair was dependent onpeptide length (see: Yaron et al., Analytical Biochemistry 95 at p. 233,col. 2, lns. 36-40). However, the later work has suggested thatefficient quenching was not so dependent on peptide length (see: Ng etal., Analytical Biochemistry, 183: at p. 54, col. 2, ln 23 to p. 55,col. 1, ln. 12; Wang et al, Tett. Lett., 31 wherein the peptide is eightamino acids in length; and Meldal et al. Analytical Biochemistry, 195 atp. 144, col. 1, lns. 33-37). It was suggested by Ng et al. that theobserved quenching in long peptides might occur by an as yetundetermined mechanism (see: Ng et al., Analytical Biochemistry 183 atp. 55, col. 1, ln 13 to col. 2, ln 7.)

Despite its name, peptide nucleic acid (PNA) is neither a peptide, anucleic acid nor is it even an acid. Peptide Nucleic Acid (PNA) is anon-naturally occurring polyamide (pseudopeptide) which can hybridize tonucleic acid (DNA and RNA) with sequence specificity (see U.S. Pat. No.5,539,082 and Egholm et al., Nature 365: 566-568 (1993)). PNAs aresynthesized by adaptation of standard peptide synthesis procedures in aformat which is now commercially available. (For a general review of thepreparation of PNA monomers and oligomers please see: Dueholm et al.,New J. Chem., 21: 19-31 (1997) or Hyrup et. al., Bioorganic & Med. Chem.4: 5-23 (1996)). Alternatively, labeled and unlabeled PNA oligomers canbe purchased (see: PerSeptive Biosystems Promotional Literature:BioConcepts, Publication No. NL612, Practical PNA, Review and PracticalPNA, Vol. 1, Iss. 2)

Being non-naturally occurring molecules, PNAs are not known to besubstrates for the enzymes which are known to degrade peptides ornucleic acids. Therefore, PNAs should be stable in biological samples,as well as have a long shelf-life. Unlike nucleic acid hybridizationwhich is very dependent on ionic strength, the hybridization of a PNAwith a nucleic acid is fairly independent of ionic strength and isfavored at low ionic strength, conditions which strongly disfavor thehybridization of nucleic acid to nucleic acid (Egholm et al., Nature, atp. 567). The effect of ionic strength on the stability and conformationof PNA complexes has been extensively investigated (Tomac et al, J. Am.Chem. Soc. 118: 5544-5552 (1996)). Sequence discrimination is moreefficient for PNA recognizing DNA than for DNA recognizing DNA (Egholmet al., Nature, at p. 566). However, the advantages in point mutationdiscrimination with PNA probes, as compared with DNA probes, in ahybridization assay appears to be somewhat sequence dependent (Nielsenet al., Anti-Cancer Drug Design 8: 53-65, (1993)). As an additionaladvantage, PNAs hybridize to nucleic acid in both a parallel andantiparallel orientation, though the antiparallel orientation ispreferred (see: Egholm et al., Nature at p. 566).

Despite the ability to hybridize to nucleic acid in a sequence specificmanner, there are many differences between PNA probes and standardnucleic acid probes. These differences can be conveniently broken downinto biological, structural, and physico-chemical differences. Asdiscussed in more detail below, these biological, structural, andphysico-chemical differences may lead to unpredictable results whenattempting to use PNA probes in applications were nucleic acids havetypically been employed. This non-equivalency of differing compositionsis often observed in the chemical arts.

With regard to biological differences, nucleic acids, are biologicalmaterials that play a central role in the life of living species asagents of genetic transmission and expression. Their in vivo propertiesare fairly well understood. PNA, on the other hand is recently developedtotally artificial molecule, conceived in the minds of chemists and madeusing synthetic organic chemistry. It has no known biological function(i.e., native (unmodified) PNA is not known to be a substrate for anypolymerase, ligase, nuclease or protease).

Structurally, PNA also differs dramatically from nucleic acid. Althoughboth can employ common nucleobases (A, C, G, T, and U), the backbones ofthese molecules are structurally diverse. The backbones of RNA and DNAare composed of repeating phosphodiester ribose and 2-deoxyribose units.In contrast, the backbones of the most common PNAs are composed onN-[2-(aminoethyl)]glycine subunits. Additionally, in PNA the nucleobasesare connected to the backbone by an additional methylene carbonylmoiety.

PNA is not an acid and therefore contains no charged acidic groups suchas those present in DNA and RNA. Because they lack formal charge, PNAsare generally more hydrophobic than their equivalent nucleic acidmolecules. The hydrophobic character of PNA allows for the possibilityof non-specific (hydrophobic/hydrophobic interactions) interactions notobserved with nucleic acids. Further, PNA is achiral, providing it withthe capability of adopting structural conformations the equivalent ofwhich do not exist in the RNA/DNA realm.

The unique structural features of PNA result in a polymer which ishighly organized in solution, particularly for purine rich polymers(see: Dueholm et al., New J. Chem., 21: 19-31 (1997) at p. 27, col. 2,lns. 6-30). Conversely, a single stranded nucleic acid is a random coilwhich exhibits very little secondary structure. Because PNA is highlyorganized, PNA should be more resistant to adopting alternativesecondary structures (e.g., a hairpin stem and/or loop).

The physico/chemical differences between PNA and DNA or RNA are alsosubstantial. PNA binds to its complementary nucleic acid more rapidlythan nucleic acid probes bind to the same target sequence. This behavioris believed to be, at least partially, due to the fact that PNA lackscharge on its backbone. Additionally, recent publications demonstratethat the incorporation of positively charged groups into PNAs willimprove the kinetics of hybridization (see: Iyer et al., J. Biol. Chem.270: 14712-14717 (1995)). Because it lacks charge on the backbone, thestability of the PNA/nucleic acid complex is higher than that of ananalogous DNA/DNA or RNA/DNA complex. In certain situations, PNA willform highly stable triple helical complexes through a process called“strand displacement”. No equivalent strand displacement processes orstructures are known in the DNA/RNA world.

Recently, the “Hybridization based screening on peptide nucleic acid(PNA) oligomer arrays” has been described wherein arrays of some 1000PNA oligomers of individual sequence were synthesized on polymermembranes (see: Weiler et al., Nucl. Acids Res. 25: 2792-2799 (1997)).Arrays are generally used, in a single assay, to generate affinitybinding (hybridization) information about a specific sequence or sampleto numerous probes of defined composition. Thus, PNA arrays may beuseful in diagnostic applications or for screening libraries ofcompounds for leads which might exhibit therapeutic utility. However,Weiler et al. note that the affinity and specificity of DNAhybridization to immobilized PNA oligomers depended on hybridizationconditions more than was expected. Moreover, there was a tendency towardnon-specific binding at lower ionic strength. Furthermore, certain verystrong binding mismatches were identified which could not be eliminatedby more stringent washing conditions. These unexpected results areillustrative of the lack of complete understanding of these newlydiscovered molecules (i.e., PNA).

In summary, because PNAs hybridize to nucleic acids with sequencespecificity, PNAs are useful candidates for investigation as substituteprobes when developing probe-based hybridization assays. However, PNAprobes are not the equivalent of nucleic acid probes in both structureor function. Consequently, the unique biological, structural, andphysico-chemical properties of PNA requires that experimentation beperformed to thereby examine whether PNAs are suitable in applicationswhere nucleic acid probes are commonly utilized.

BRIEF SUMMARY OF THE INVENTION

Numerous PNA polymers were examined in an attempt to prepare a PNAMolecular Beacon. The applicants have determined that all PNA oligomersthey examined, which contained donor and acceptor moieties located atopposite ends of the polymer, exhibited a low inherent background and adetectable increase in signal upon binding of the probe to a targetsequence. Very surprisingly, these characteristic properties of anucleic acid Molecular Beacon were observed whether or not the PNAoligomer was suitable for adopting a hairpin stem and loop structure ina manner commensurate with the design of the nucleobase sequence. Forexample, in hybridization assay analysis, the PNA oligomers (originallydesigned as control oligomers) which do not possess any arm segmentssuitable for creating a hairpin, exhibited a signal (PNA oligomer boundto target sequence) to noise (no target sequence present) ratio which isapproximately half that observed for PNAs which compriseself-complementary nucleobase sequences.

This invention is directed to Linear Beacons. A Linear Beacon,efficiently transfers energy between the donor and acceptor moietieslinked to the probe in the absence of target sequence whether or not, bydesign, it comprises self complementary nucleobase sequence. Uponhybridization to a target sequence, the efficiency of energy transferbetween donor and acceptor moieties of the Linear Beacon is altered suchthat detectable signal from at least one moiety can be used to monitoror quantitate occurrence of the hybridization event. We refer to theseprobes as Linear Beacons to distinguish them from the hairpin structurestypically associated with nucleic acid Molecular Beacons. Nevertheless,applicants do not intend to imply that these probes lack a secondarystructure since the literature teaches that PNAs can be highly organizedin solution (see: Dueholm et al., New J. Chem., 21: 19-31 (1997 at p.27, col. 2, lns. 6-30).

The Linear Beacons of this invention possess several properties whichare unique and not predicable. For example, applicants demonstrate thatthe efficiency of energy transfer between donor and acceptor moieties ofa Linear Beacon is substantially independent of length since essentiallythe same noise (see: Example 17 of this specification) and signal tonoise ratio (see: Example 18 of this specification) was observed foroligomers of 11-17 subunits in length. This was a very surprising resultsince the intramolecular quenching of suitably labeled nucleic acidoligomers is very dependent on the length of the probe (see: Backgroundand the data presented in Example 17 of this specification).

Additionally, applicants have demonstrated that the efficiency ofquenching of a Linear Beacon is neither sequence dependent, norsubstantially dependent on the spectral overlap of the donor andacceptor moieties (see: Examples 17, 18 and 21 of this specification).Specifically, the majority of PNA probes which were prepared comprise afluorescein donor moiety and a dabcyl quencher (acceptor) moiety. Thoughthis donor/acceptor combination is not an ideal FRET pair since theemission of the donor fluorescein moiety does not have a high degree ofspectral overlap with the absorption of the acceptor dabcyl moiety, thequenching observed by applicants is nevertheless substantial in allconstructs. Furthermore, Linear Beacons comprising the Cy3/dabcyldonor/acceptor pair, respectively, were observed to exhibit both a noiseand a signal to noise ratio which was similar to that seen for thefluorescein/dabcyl system despite there being substantially lessspectral overlap between Cy3 and dabcyl (see: Examples 17, 18 and 21 ofthis specification). Consequently, the data compiled by applicantssurprisingly demonstrates that Linear Beacons need not comprise optimalFRET pairs to be operable. Consequently, the data suggests that directcontact is the primary, but likely not the only, mode of energy transfersince spectral overlap is a requirement for FRET but is not required fornon-FRET energy transfer. Furthermore, the data suggests that regardlessof probe length, the fluorophore and quencher moieties of a LinearBeacon are similarly situated to thereby achieve a degree of quenchingwhich is fairly independent of probe length or nucleobase sequence.

Applicants have likewise investigated what effect varying ionic strengthand particularly the presence or absence that magnesium has on probenoise and signal to noise ratios. Again, PNAs were found to exhibitnoise and signal to noise ratios which were substantially independent ofdifferences in ionic strength or presence or absence of magnesiumwhereas the properties of DNA probes of similar length and labelingconfiguration were dependent on variations in ionic strength and/orhighly dependent on the presence or absence of magnesium.

In summary, it has also been observed by applicants that the noise andsignal to noise ratio for Linear Beacons is substantially independent oflength of subunits which separate donor and acceptor moieties, ionicstrength of the environment or the presence or absence of magnesium.When considered as a whole, these results were very unexpected in lightof prior art teachings. Consequently, applicants data demonstrates aclear non-equivalency of structure and function between nucleic acid andPNA probes of similar length and labeling configurations. It followsthat the novel methods, kits and compositions of this invention compriseLinear Beacons which possess unique and surprising properties.

In one embodiment, this invention is directed to a Linear Beacon.Generally, a Linear Beacon is a polymer which at a minimum comprises atleast one linked donor moiety and at least one linked acceptor moietywherein said donor and acceptor moieties are separated by a at least aportion of a probing nucleobase sequence wherein the probing nucleobasesequence is suitable for hybridization to a complementary orsubstantially complementary target sequence, under suitablehybridization conditions. By design, a Linear Beacon does not form ahairpin stem. The Linear Beacon is further characterized in that theefficiency of transfer of energy between said donor and acceptormoieties, when the polymer is solvated in aqueous solution, issubstantially independent of at least two variable factors selected fromthe group consisting of length of subunits which separate donor andacceptor moieties, spectral overlap of the donor moiety and the acceptormoiety, presence or absence of magnesium in the aqueous solution and theionic strength of the aqueous solution. Preferably the Linear Beacon isfurther characterized in that the efficiency of transfer of energybetween said donor and acceptor moieties is substantially independent ofat least three variable factors and most preferably substantiallyindependent of all four variable factors.

In a preferred embodiment, a Linear Beacon is a polymer comprising PNAsubunits which, at a minimum, consists of a probing nucleobase sequencehaving a first and second end. The probing nucleobase sequence iscomplementary or substantially complementary to a target sequence ofinterest. At least one donor moiety is linked to one of the first orsecond ends of the probing nucleobase sequence; and at least oneacceptor moiety is linked to the other one of the first or second end ofthe probing nucleobase sequence. One or more spacer or linker moietiesmay be used to link the donor and acceptor moieties to the respectiveends of the probing nucleobase sequence.

In another embodiment, this invention is related to a method for thedetection, identification or quantitation of a target sequence in asample. The method comprises contacting the sample with a Linear Beaconand then detecting, identifying or quantitating the change in detectablesignal associated with at least one donor or acceptor moiety of theprobe whereby the change in detectable signal is used to determine thepresence, absence or amount of target sequence present in the sample ofinterest. The measurable change in detectable signal of at least onedonor or acceptor moiety of the probe can be used to determine thepresence, absence or amount of target sequence present in the sample ofinterest since applicants have demonstrated that the efficiency ofenergy transfer between donor and acceptor moieties is altered byhybridization of the Linear Beacons to their intended target sequences,under suitable hybridization conditions. Accurate quantitation can beachieved by correcting for signal generated by any unhybridized LinearBeacon. Consequently, the Linear Beacons of this invention areparticularly well suited for the detection, identification orquantitation of target sequences in closed tube assays and particularlyasymmetric PCR assays (see: Example 19 of this specification). Becausethe Linear Beacons are not known to be degraded by enzymes, the LinearBeacons are also particularly well suited for detection, identificationor quantitation of target sequences in cells, tissues or organisms,whether living or not (see: Example 20 of this specification).

In still another embodiment, this invention is related to kits suitablefor performing an assay which detects the presence, absence or number ofa target sequences in a sample. The kits of this invention comprise oneor more Linear Beacons and other reagents or compositions which areselected to perform an assay or otherwise simplify the performance of anassay.

In yet another embodiment, this invention also is directed to an arraycomprising two or more support bound Linear Beacons suitable fordetecting, identifying or quantitating a target sequence of interest.Arrays of Linear Beacons are convenient because they provide a means torapidly interrogate numerous samples for the presence of one or moretarget sequences of interest in real time without using a secondarydetection system.

The methods, kits and compositions of this invention are particularlyuseful for the detection of target sequences of organisms which may befound in food, beverages, water, pharmaceutical products, personal careproducts, dairy products or environmental samples. The analysis ofpreferred beverages include soda, bottled water, fruit juice, beer, wineor liquor products. Additionally, the methods, kits and compositionswill be particularly useful for the analysis of raw materials,equipment, products or processes used to manufacture or store food,beverages, water, pharmaceutical products, personal care products dairyproducts or environmental samples.

Whether support bound or in solution, the methods, kits and compositionsof this invention are particularly useful for the rapid, sensitive,reliable and versatile detection of target sequences which areparticular to organisms which might be found in clinical environments.Consequently, the methods, kits and compositions of this invention willbe particularly useful for the analysis of clinical specimens orequipment, fixtures or products used to treat humans or animals. Forexample, the assay may be used to detect a target sequence which isspecific for a genetically based disease or is specific for apredisposition to a genetically based disease. Non-limiting examples ofdiseases include, β-Thalassemia, sickle cell anemia, Factor-V Leiden,cystic fibrosis and cancer related targets such as p53, p10, BRC-1 andBRC-2.

In still another embodiment, the target sequence may be related to achromosomal DNA, wherein the detection, identification or quantitationof the target sequence can be used in relation to forensic techniquessuch as prenatal screening, paternity testing, identity confirmation orcrime investigation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graphical illustration of fluorescence vs. temperature datafor PNA probes which exhibit a Type A Fluorescent Thermal Profile.

FIGS. 1B1, 1B2 and 1B3 are graphical illustrations of fluorescence vs.temperature data for PNA probes which exhibit a Type B FluorescentThermal Profile.

FIG. 1C is a graphical illustration of fluorescence vs. temperature datafor PNA probes which exhibit a Type C Fluorescent Thermal Profile.

FIGS. 2A1, 2A2 and 2A3 are a graphical illustration of fluorescence vs.time data for PNA probes which exhibit a Type A Hybridization Profile.

FIG. 2B is a graphical illustration of fluorescence vs. time data forPNA probes which exhibit a Type B Hybridization Profile.

FIG. 2C is a graphical illustration of fluorescence vs. time data forPNA probes which exhibit a Type C Hybridization Profile.

FIG. 3 is a graphical illustration of noise (background fluorescence)data for DNA and PNA probes of different lengths and labelingconfigurations.

FIGS. 4A, 4B, 4C, 4D and 4E are graphical illustrations of signal tonoise data for PNA and DNA probes of 11 and 15 subunits in length.

FIG. 5 is a digital image of two eppendorf tubes each containingcontents of a reaction which underwent 45 cycles of PCR and containing aLinear Beacon.

FIGS. 6A and 6B are digital images of sample slides containing E. coli,P. aeruginosa or B. subtilis bacteria which were treated with LinearBeacons and propidium iodide wherein the Linear Beacons comprise probingnucleobase sequence specific to either P. aeruginosa (FIG. 6A) or B.subtilis (FIG. 6B). The images were obtained using a fluorescencemicroscope and commercially available light filters fitted to themicroscope and the camera respectively. In both Figures, Panels I, IIIand IV are the red images obtained using a red microscope and camerafilter wherein the propidium iodide stained cells are visible. In bothFigures, Panels II, IV and VI are the green images obtained using agreen microscope and camera filter.

FIGS. 7A and 7B are graphical representations of data compiled for noiseand signal to noise ratios for a Cy3 labeled 15-mer PNA probe having ascrambled nucleobase sequence.

FIG. 8 is a graphical illustration of hybridization assay signal tonoise ratios for probes listed in Table 1A.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

As used herein, the term “nucleobase” shall include those naturallyoccurring and those non-naturally occurring heterocyclic moietiescommonly known to those who utilize nucleic acid technology or utilizepeptide nucleic acid technology to thereby generate polymers which cansequence specifically bind to nucleic acids.

As used herein, the term “nucleobase sequence” is any segment of apolymer which comprises nucleobase containing subunits. Non-limitingexamples of suitable polymers or polymers segments includeoligonucleotides, oligoribonucleotides, peptide nucleic acids andanalogs or chimeras thereof.

As used herein, the term “target sequence” is any sequence ofnucleobases in a polymer which is sought to be detected. The “targetsequence” may comprise the entire polymer or may be a subsequence of thenucleobase sequence which is unique to the polymer of interest. Withoutlimitation, the polymer comprising the “target sequence” may be anucleic acid, a peptide nucleic acid, a chimera, a linked polymer, aconjugate or any other polymer comprising substituents (e.g.,nucleobases) to which the Linear Beacons of this invention may bind in asequence specific manner.

As used herein, the term “peptide nucleic acid” or “PNA” shall bedefined as any oligomer, linked polymer or chimeric oligomer, comprisingtwo or more PNA subunits (residues), including any of the compoundsreferred to or claimed as peptide nucleic acids in U.S. Pat. Nos.5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 or5,786,571 (all of which are herein incorporated by reference). The term“Peptide Nucleic Acid” or “PNA” shall also apply to those nucleic acidmimics described in the following publications: Diderichsen et al, Tett.Lett. 37:475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett.7:637-627 (1997); Jordan et al, Bioorg. Med. Chem. Lett. 7:687-690(1997); Krotz et al., Tett. Lett. 36:6941-6944 (1995); Lagriffoul etal., Bioorg. Med. Chem. Lett. 4:1081-1082 (1994); Lowe et al., J. Chem.Soc. Perkin Trans. 1, (1997) 1:539-546; Lowe et al., J. Chem. Soc.Perkin Trans. 11:547-554 (1997); Lowe et al., J. Chem. Soc. PerkinTrans. 11:555-560 (1997); and Petersen et al, Bioorg. Med. Chem. Lett.6:793-796 (1996).

In preferred embodiments, a PNA is a polymer comprising two or more PNAsubunits of the formula:

wherein, each J is the same or different and is selected from the groupconsisting of H, R¹, OR¹, SR¹, NHR¹, NR¹ ₂, F, Cl, Br and I. Each K isthe same or different and is selected from the group consisting of O, S,NH and NR¹. Each R¹ is the same or different and is an alkyl grouphaving one to five carbon atoms which may optionally contain aheteroatom or a substituted or unsubstituted aryl group. Each A isselected from the group consisting of a single bond, a group of theformula; —(CJ₂)_(s)- and a group of the formula; —(CJ₂)_(s)C(O)—,wherein, J is defined above and each s is an integer from one to five.The integer t is 1 or 2 and the integer u is 1 or 2. Each L is the sameor different and is independently selected from the group consisting ofJ, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturallyoccurring nucleobase analogs, other non-naturally occurring nucleobases,substituted and unsubstituted aromatic moieties, biotin and fluorescein.In the most preferred embodiment, a PNA subunit consists of a naturallyoccurring or non-naturally occurring nucleobase attached to the azanitrogen of the N-[2-(aminoethyl)]glycine backbone through a methylenecarbonyl linkage.

2. Detailed Description I. GENERAL

PNA Synthesis:

Methods for the chemical assembly of PNAs are well known (see: U.S. Pat.Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 or5,786,571 (all of which are herein incorporated by reference). Chemicalsand instrumentation for the support bound automated chemical assembly ofPeptide Nucleic Acids are now commercially available. Chemical assemblyof a PNA is analogous to solid phase peptide synthesis, wherein at eachcycle of assembly the oligomer possesses a reactive alkyl amino terminuswhich is condensed with the next synthon to be added to the growingpolymer. Because standard peptide chemistry is utilized, natural andnon-natural amino acids are routinely incorporated into a PNA oligomer.Because a PNA is a polyamide, it has a C-terminus (carboxyl terminus)and an N-terminus (amino terminus). For the purposes of the design of ahybridization probe suitable for antiparallel binding to the targetsequence (the preferred orientation), the N-terminus of the probingnucleobase sequence of the PNA probe is the equivalent of the5′-hydroxyl terminus of an equivalent DNA or RNA oligonucleotide.

Labels:

The labels attached to the Linear Beacons of this invention comprise aset (hereinafter “Beacon Set(s)”) of energy transfer moieties comprisingat least one energy donor and at least one energy acceptor moiety.Typically, the Beacon Set will include a single donor moiety and asingle acceptor moiety. Nevertheless, a Beacon Set may contain more thanone donor moiety and/or more than one acceptor moiety. The donor andacceptor moieties operate such that one or more acceptor moietiesaccepts energy transferred from the one or more donor moieties orotherwise quench signal from the donor moiety or moieties.

Preferably the donor moiety is a fluorophore. Preferred fluorophores arederivatives of fluorescein, derivatives of bodipy,5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), derivativesof rhodamine, Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, texas red and itsderivatives. Though the previously listed fluorophores might alsooperate as acceptors, preferably, the acceptor moiety is a quenchermoiety. Preferably, the quencher moiety is a non-fluorescent aromatic orheteroaromatic moiety. The preferred quencher moiety is4-((-4-(dimethylamino)phenyl)azo) benzoic acid (dabcyl).

Transfer of energy may occur through collision of the closely associatedmoieties of a Beacon Set or through a nonradiative process such asfluorescence resonance energy transfer (FRET). For FRET to occur,transfer of energy between donor and acceptor moieties of a Beacon Setrequires that the moieties be close in space and that the emissionspectrum of a donor(s) have substantial overlap with the absorptionspectrum of the acceptor(s) (see: Yaron et al. Analytical Biochemistry,95: 228-235 (1979) and particularly page 232, col. 1 through page 234,col. 1). Alternatively, collision mediated (radiationless) energytransfer may occur between very closely associated donor and acceptormoieties whether or not the emission spectrum of a donor moiety(ies) hasa substantial overlap with the absorption spectrum of the acceptormoiety(ies) (see: Yaron et al., Analytical Biochemistry, 95: 228-235(1979) and particularly page 229, col. 1 through page 232, col. 1). Thisprocess is referred to as intramolecular collision since it is believedthat quenching is caused by the direct contact of the donor and acceptormoieties (see: Yaron et al.). As applicants have demonstrated, the donorand acceptor moieties attached to the Linear Beacons of this inventionneed not have a substantial overlap between the emission of the donormoieties and the absorbance of the acceptor moieties. Without intendingto be bound to this hypothesis, this data suggests that collision orcontact operates as the primary mode of quenching in Linear Beacons.

Detecting Energy Transfer:

Because the efficiency of both collision mediated and nonradiativetransfer of energy between the donor and acceptor moieties of a BeaconSet is directly dependent on the proximity of the donor and acceptormoieties, detection of hybrid formation of a Linear Beacon with a targetsequence can be monitored by measuring at least one physical property ofat least one member of the Beacon Set which is detectably different whenthe hybridization complex is formed as compared with when the LinearBeacon exists in the absence of target sequence. We refer to thisphenomenon as the self-indicating property of Linear Beacons. Thischange in detectable signal shall result from the change in efficiencyof energy transfer between the donor and acceptor which results fromhybridization of the Linear Beacon. Preferably, the means of detectionwill involve measuring fluorescence of a donor or acceptor fluorophoreof a Beacon Set. Most preferably, the Beacon Set will comprise at leastone donor fluorophore and at least one acceptor quencher such that thefluorescence of the donor fluorophore is will be used to detect,identify or quantitate hybridization.

PNA Labeling:

Chemical labeling of a PNA is analogous to peptide labeling. Because thesynthetic chemistry of assembly is essentially the same, any methodcommonly used to label a peptide may be used to label a PNA. Typically,the N-terminus of the polymer is labeled by reaction with a moietyhaving a carboxylic acid group or activated carboxylic acid group. Oneor more spacer moieties can optionally be introduced between thelabeling moiety and the probing nucleobase sequence of the oligomer.Generally, the spacer moiety is incorporated prior to performing thelabeling reaction. However, the spacer may be embedded within the labeland thereby be incorporated during the labeling reaction.

Typically the C-terminal end of the probing nucleobase sequence islabeled by first condensing a labeled moiety with the support upon whichthe PNA is to be assembled. Next, the first synthon of the probingnucleobase sequence can be condensed with the labeled moiety.Alternatively, one or more spacer moieties can be introduced between thelabeled moiety and the oligomer (e.g., 8-amino-3,6-dioxaoctanoic acid).Once the Linear Beacon is completely assembled and labeled, it iscleaved from the support deprotected and purified using standardmethodologies.

The labeled moiety could be a lysine derivative wherein the 6-aminogroup is modified with a donor or acceptor moiety. For example the labelcould be a fluorophore such as 5(6)-carboxyfluorescein or a quenchermoiety such as 4-((4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).Condensation of the lysine derivative with the synthesis support wouldbe accomplished using standard condensation (peptide) chemistry. Theα-amino group of the lysine derivative would then be deprotected and theprobing nucleobase sequence assembly initiated by condensation of thefirst PNA synthon with the α-amino group of the lysine amino acid. Asdiscussed above, a spacer moiety could optionally be inserted betweenthe lysine amino acid and the first PNA synthon by condensing a suitablespacer (e.g., Fmoc-8-amino-3,6-dioxaoctanoic acid) with the lysine aminoacid prior to condensation of the first PNA synthon of the probingnucleobase sequence.

Alternatively, a functional group on the assembled, or partiallyassembled, polymer is labeled with a donor or acceptor moiety while itis still support bound. This method requires that an appropriateprotecting group be incorporated into the oligomer to thereby yield areactive functional group to which the donor or acceptor moiety islinked but has the advantage that the label (e.g., dabcyl or afluorophore) can be attached to any position within the polymerincluding within the probing nucleobase sequence. For example, the□-amino group of a lysine could be protected with a4-methyl-triphenylmethyl (Mtt), a 4-methoxy-triphenylmethyl (MMT) or a4,4′-dimethoxytriphenylmethyl (DMT) protecting group. The Mtt, MMT orDMT groups can be removed from PNA (assembled using commerciallyavailable Fmoc PNA monomers and polystyrene support having a PAL linker;PerSeptive Biosystems, Inc., Framingham, Mass.) by treatment of theresin under mildly acidic conditions. Consequently, the donor oracceptor moiety can then be condensed with the ε-amino group of thelysine amino acid. After complete assembly and labeling, the polymer isthen cleaved from the support, deprotected and purified using well knownmethodologies.

By still another method, the donor or acceptor moiety is attached to thepolymer after it is fully assembled and cleaved from the support. Thismethod is preferable where the label is incompatible with the cleavage,deprotection or purification regimes commonly used to manufacture theoligomer. By this method, the PNA will generally be labeled in solutionby the reaction of a functional group on the polymer and a functionalgroup on the label. Those of ordinary skill in the art will recognizethat the composition of the coupling solution will depend on the natureof oligomer and the donor or acceptor moiety. The solution may compriseorganic solvent, water or any combination thereof. Generally, theorganic solvent will be a polar non-nucleophilic solvent. Non limitingexamples of suitable organic solvents include acetonitrile,tetrahydrofuran, dioxane, methyl sulfoxide and N,N′-dimethylformamide.

Generally the functional group on the polymer to be labeled will be anamine and the functional group on the label will be a carboxylic acid oractivated carboxylic acid. Non-limiting examples of activated carboxylicacid functional groups include N-hydroxysuccinimidyl esters. In aqueoussolutions, the carboxylic acid group of either of the PNA or label(depending on the nature of the components chosen) can be activated witha water soluble carbodiimide. The reagent,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), is acommercially available reagent sold specifically for aqueous amideforming condensation reactions.

Generally, the pH of aqueous solutions will be modulated with a bufferduring the condensation reaction. Preferably, the pH during thecondensation is in the range of 4-10. When an arylamine is condensedwith the carboxylic acid, preferably the pH is in the range of 4-7. Whenan alkylamine is condensed with a carboxylic acid, preferably the pH isin the range of 7-10. Generally, the basicity of non-aqueous reactionswill be modulated by the addition of non-nucleophilic organic bases.Non-limiting examples of suitable bases include N-methylmorpholine,triethylamine and N,N-diisopropylethylamine. Alternatively, the pH ismodulated using biological buffers such as(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid) (HEPES) or4-morpholineethane-sulfonic acid (MES) or inorganic buffers such assodium bicarbonate.

Spacer/Linker Moieties:

Generally, spacers are used to minimize the adverse effects that bulkylabeling reagents might have on hybridization properties of LinearBeacons. Linkers typically induce flexibility and randomness into theLinear Beacon or otherwise link two or more nucleobase sequences of aprobe. Preferred spacer/linker moieties for probes of this inventionconsist of one or more aminoalkyl carboxylic acids (e.g., aminocaproicacid) the side chain of an amino acid (e.g., the side chain of lysine orornithine) natural amino acids (e.g., glycine), aminooxyalkylacids(e.g., 8-amino-3,6-dioxaoctanoic acid), alkyl diacids (e.g., succinicacid) or alkyloxy diacids (e.g., diglycolic acid). The spacer/linkermoieties may also be designed to enhance the solubility of the LinearBeacon.

Preferably, a spacer/linker moiety comprises one or more linkedcompounds having the formula: —Y—(O_(m)—(CW₂)_(n))_(o)-Z-. The group Yhas the formula: a single bond, —(CW₂)_(p)—, —C(O)(CW₂)_(p)—,—C(S)(CW₂)_(p)— and —S(O₂)(CW₂)_(p). The group Z has the formula NH,NR², S or O. Each W is independently H, R², —OR², F, Cl, Br or I;wherein, each R² is independently selected from the group consisting of:—CX₃, —CX₂CX₃, —CX₂CX₂CX₃, —CX₂CX(CX₃)₂, and —C(CX₃)₃. Each X isindependently H, F, Cl, Br or I. Each m is independently 0 or 1. Each n,o and p are independently integers from 0 to 10.

Chimeric Oligomer:

A chimeric oligomer comprises two or more linked subunits which areselected from different classes of subunits. For example, a PNA/DNAchimera would comprise at least two PNA subunits linked to at least one2′-deoxyribonucleic acid subunit (For methods and compositions relatedto PNA/DNA chimera preparation See: WO96/40709). The component subunitsof the chimeric oligomers are selected from the group consisting of PNAsubunits, DNA subunits, RNA subunits and analogues thereof.

Linked Polymer:

A linked polymer comprises two or more nucleobase sequences which arelinked by a linker. The nucleobase sequences which are linked to formthe linked polymer are selected from the group consisting of anoligodeoxynucleotide, an oligoribonucleotide, a peptide nucleic acid anda chimeric oligomer. The PNA probes of this invention include linkedpolymers wherein the probing nucleobase sequence is linked to one ormore additional oligodeoxynucleotide, oligoribonucleotide, peptidenucleic acid or chimeric oligomers.

Hybridization Conditions/Stringency:

Those of ordinary skill in the art of nucleic acid hybridization willrecognize that factors commonly used to impose or control stringency ofhybridization include formamide concentration (or other chemicaldenaturant reagent), salt concentration (i.e., ionic strength),hybridization temperature, detergent concentration, pH and the presenceor absence of chaotropes. Optimal stringency for a probing nucleobasesequence/target sequence combination is often found by the well knowntechnique of fixing several of the aforementioned stringency factors andthen determining the effect of varying a single stringency factor. Thesame stringency factors can be modulated to thereby control thestringency of hybridization of Linear Beacons to target sequences,except that the hybridization of a PNA is fairly independent of ionicstrength. Optimal stringency for an assay may be experimentallydetermined by examination of each stringency factor until the desireddegree of discrimination is achieved.

Probing Nucleobase Sequence:

The probing nucleobase sequence of a Linear Beacon is the sequencerecognition portion of the construct. Therefore, the probing nucleobasesequence is designed to hybridize to at least a portion of the targetsequence. Preferably the probing nucleobase sequence hybridizes to theentire target sequence. The probing nucleobase sequence is anon-polynucleotide and preferably the probing nucleobase sequence iscomposed exclusively of PNA subunits. The subunit length of the probingnucleobase sequence will therefore generally be chosen such that astable complex is formed between the Linear Beacon and the targetsequence sought to be detected, under suitable hybridization conditions.The probing nucleobase sequence of a PNA oligomer, suitable for thepractice of this invention, will generally have a length of between 5and 30 PNA subunits. Preferably, the probing nucleobase sequence will be8 to 18 subunits in length. Most preferably, the probing nucleobasesequence will be 11-17 subunits in length. The probing nucleobasesequence of Linear Beacons will generally have a nucleobase sequencewhich is complementary to the target sequence. Alternatively, asubstantially complementary probing sequence might be used since it hasbeen demonstrated that greater sequence discrimination can be obtainedwhen utilizing probes wherein there exists a single point mutation (basemismatch) between the probing nucleobase sequence and the targetsequence (see: Guo et al., Nature Biotechnology 15: 331-335 (1997), Guoet al., WO97/46711; and Guo et al., U.S. Pat. No. 5,780,233, hereinincorporated by reference).

Blocking Probes:

Blocking probes are PNA or nucleic acid probes which can be used tosuppress the binding of the probing nucleobase sequence of a probe to ahybridization site which is unrelated or closely related to the targetsequence (see: Coull et al., PCT/US97/21845, a.k.a. WO98/24933).Generally, the blocking probes suppress the binding of the probingnucleobase sequence to closely related non-target sequences because theblocking probe hybridizes to the non-target sequence to form a morethermodynamically stable complex than is formed by hybridization betweenthe probing nucleobase sequence and the non-target sequence. Thus,blocking probes are typically unlabeled probes used in an assay tothereby suppress non-specific signal. Because they are usually designedto hybridize to closely related non-target sequence sequences, typicallya set of two or more blocking probes will be used in an assay to therebysuppress non-specific signal from non-target sequences which could bepresent and interfere with the performance of the assay.

II. PREFERRED EMBODIMENTS OF THE INVENTION

Linear Beacon Probes:

Linear Beacons are disclosed which are suitable for facilitating energytransfer between donor and acceptor moieties when the probe is nothybridized to its target sequence. However, hybridization of the probeto its target sequence will alter the efficiency of energy transferbetween donor and acceptor moieties and thereby result in a measurablechange in signal associated with at least one member of the Beacon Set.

Generally, a Linear Beacon is a polymer which at a minimum comprises atleast one linked donor moiety and at least one linked acceptor moietywherein said donor and acceptor moieties are separated by a at least aportion of a probing nucleobase sequence wherein the probing nucleobasesequence is suitable for hybridization to a complementary orsubstantially complementary target sequence, under suitablehybridization conditions. By design, a Linear Beacon does not form ahairpin stem. Preferably the donor and acceptor moieties are linked atopposite ends of the probing nucleobase sequence. The Linear Beacon isfurther characterized in that the efficiency of transfer of energybetween said donor and acceptor moieties, when the polymer is solvatedin aqueous solution, is substantially independent of at least twovariable factors selected from the group consisting of length ofsubunits which separate donor and acceptor moieties, spectral overlap ofthe donor moiety and the acceptor moiety, presence or absence ofmagnesium in the aqueous solution and the ionic strength of the aqueoussolution. Preferably the Linear Beacon is further characterized in thatthe efficiency of transfer of energy between said donor and acceptormoieties is substantially independent of at least three variable factorsand most preferably substantially independent of all four variablefactors.

In a preferred embodiment, the Linear Beacon is a polymer which, at aminimum, consists of a probing nucleobase sequence having a first andsecond end. The probing nucleobase sequence is complementary orsubstantially complementary to a target sequence of interest. At leastone donor moiety is linked to one of the first or second ends of theprobing nucleobase sequence; and at least one acceptor moiety is linkedto the other one of the first or second end of the probing nucleobasesequence. One or more spacer or linker moieties may be used to link thedonor and acceptor moieties to the respective ends of the probingnucleobase sequence. In a most preferred embodiment, the Linear Beaconis a PNA oligomer.

Linear Beacons may comprise only a probing nucleobase sequence (aspreviously described herein) and linked donor and acceptor moieties of aBeacon Set or may optionally comprise additional linked moieties.Non-limiting examples of additional linked moieties include otherlabels, linkers, spacers, natural or non-natural amino acids, peptides,proteins, nucleic acids, enzymes and/or one or more other subunits ofPNA, DNA or RNA. Additional moieties may be functional or non-functionalin an assay. Generally however, additional moieties will be selected tobe functional within the design of the assay in which the Linear Beaconsis to be used. The Linear Beacons of this invention may optionally beimmobilized to a surface.

As a non-limiting example, a Linear Beacon of this invention maycomprise a nucleic acid linked to a probing nucleobase sequence whereinthe nucleic acid might hybridize to a second target sequence. As asecond non-limiting example, a Linear Beacon may comprise an enzymelinked to a probing nucleobase sequence wherein the enzyme might be usedin a secondary detection scheme. A third non-limiting example of aLinear Beacon could comprise an antibody linked to the probingnucleobase sequence wherein the antibody might be used in a secondarydetection scheme. As still a fourth non-limiting example, a LinearBeacon might comprise one or more spacer moieties linked to a probingnucleobase sequence wherein the one or more spacer moieties are used totether the Linear Beacon to a support.

Unique Features of Linear Beacons:

There are many differences between prior art nucleic acid constructs andthe Linear Beacons of this invention. For example, nucleic acidconstructs comprise a polynucleotide backbone whereas the Linear Beaconsof this invention comprise a probing nucleobase sequences which is otherthan a polynucleotide. In a preferred embodiment, Linear Beaconscomprised of PNA exhibit all of the favorable properties of PNA such asresistance to nuclease degradation, salt independent sequencehybridization to complementary nucleic acids and extremely rapidhybridization kinetics.

Additionally, the transfer of energy between donor and acceptor moietiesin a Linear Beacon is substantially independent on the presence orabsence of magnesium, the ionic strength of the probe environment andthe nucleobase sequence of the probe (see: Examples 17, 18 and 21 ofthis specification). More surprisingly, the efficiency of transfer ofenergy between donor and acceptor moieties of a Beacon Set issubstantially independent of the length of subunits which separate donorand acceptor moieties whereas the energy transfer between moietieswithin nucleic acids having the same nucleobase sequence and labelingconfiguration exhibit a substantial dependence on probe length (see:Examples 17 and 18 of this specification).

Most surprisingly, Linear Beacons operate whether or not the donor andacceptor moieties exhibit substantial overlap of the emission spectrumof the donor moiety and the absorbance spectrum of acceptor moiety (see:Examples 17, 18 and 21 of this specification). Without intending to bebound to this hypothesis, this data suggests that collision or contactoperates as the primary mode of energy transfer in Linear Beacons ascompared with nucleic acids wherein FRET has been described as theprimary source for energy transfer between donor and acceptor moieties.

Additional advantages of Linear Beacons include ease of synthesis ascompared with constructs which comprise additional subunits to form armsegments. Additionally, the data compiled by applicants demonstratesthat Linear Beacons hybridize faster than constructs which compriseadditional subunits to form arm segments (see: Examples 15 and 16 ofthis specification).

Probe Sets:

In another embodiment, this invention is directed to sets of LinearBeacons suitable for detecting or identifying the presence, absence oramount of two or more different target sequences which might be presentin a sample. The characteristics of Linear Beacons suitable for thedetection, identification or quantitation of target sequences have beenpreviously described herein. The grouping of Linear Beacons within setscharacterized for specific detection of two or more target sequences isa preferred embodiment of this invention.

Probe sets of this invention shall comprise at least one Linear Beaconbut need not comprise only Linear Beacons. For example, probe sets ofthis invention may comprise mixtures of Linear Beacons, other PNA probesand/or nucleic acid probes, provided however that a set comprises atleast one Linear Beacon as described herein. In preferred embodiments,at least one probe of the set is a blocking probe, as defined herein.

Immobilization of a Linear Beacon to a Surface:

One or more Linear Beacons may optionally be immobilized to a surface.In one embodiment, the probe can be immobilized to the surface using thewell known process of UV-crosslinking. Alternatively, the PNA oligomeris synthesized on the surface in a manner suitable for deprotection butnot cleavage from the synthesis support.

Preferably, the probe is covalently linked to a surface by the reactionof a suitable functional groups on the probe and support. Functionalgroups such as amino groups, carboxylic acids and thiols can beincorporated in a Linear Beacon by extension of one of the termini withsuitable protected moieties (e.g., lysine, glutamic acid and cystine).When extending the terminus, one functional group of a branched aminoacid such as lysine can be used to incorporate the donor or acceptorlabel at the appropriate position in the polymer (see: Section entitled“PNA Labeling”) while the other functional group of the branch is usedto optionally further extend the polymer and immobilize it to a surface.

Methods for the attachment of probes to surfaces generally involve thereaction of a nucleophilic group, (e.g., an amine or thiol) of the probeto be immobilized, with an electrophilic group on the support to bemodified. Alternatively, the nucleophile can be present on the supportand the electrophile (e.g., activated carboxylic acid) present on theLinear Beacon. Because native PNA possesses an amino terminus, a PNAwill not necessarily require modification to thereby immobilize it to asurface (see: Lester et al., Poster entitled “PNA Array Technology”).

Conditions suitable for the immobilization of a PNA to a surface willgenerally be similar to those conditions suitable for the labeling of aPNA (see: subheading “PNA Labeling”). The immobilization reaction isessentially the equivalent of labeling the PNA whereby the label issubstituted with the surface to which the PNA probe is to be covalentlyimmobilized.

Numerous types of surfaces derivatized with amino groups, carboxylicacid groups, isocyantes, isothiocyanates and malimide groups arecommercially available. Non-limiting examples of suitable surfacesinclude membranes, glass, controlled pore glass, polystyrene particles(beads), silica and gold nanoparticles.

When immobilized to a surface, energy transfer between moieties of aBeacon Set will occur in the Linear Beacon. Upon hybridization to atarget sequence under suitable hybridization conditions, the location onthe surface where the Linear Beacon (of known sequence) is attached willgenerate detectable signal based on the measurable change in signal ofat least one member of the Beacon Set of the immobilized Linear Beacon.Consequently, the intensity of the signal on the surface can be used todetect, identify or quantitate the presence or amount of a targetsequence in a sample which contacts the surface to which the LinearBeacon is immobilized. In a preferred embodiment, detection of surfacefluorescence will be used to detect hybridization to a target sequence.

Detectable and Independently Detectable Moieties/Multiplex Analysis:

In preferred embodiments of this invention, a multiplex hybridizationassay is performed. In a multiplex assay, numerous conditions ofinterest are simultaneously examined. Multiplex analysis relies on theability to sort sample components or the data associated therewith,during or after the assay is completed. In preferred embodiments of theinvention, distinct independently detectable moieties are used to labelthe different Linear Beacons of a set. The ability to differentiatebetween and/or quantitate each of the independently detectable moietiesprovides the means to multiplex a hybridization assay because the datawhich correlates with the hybridization of each of the distinctly(independently) labeled Linear Beacons to a target sequence can becorrelated with the presence, absence or quantity of the target sequencesought to be detected in a sample. Consequently, the multiplex assays ofthis invention may be used to simultaneously detect the presence,absence or amount of one or more target sequences which may be presentin the same sample in the same assay. Preferably, independentlydetectable fluorophores will be used as the independently detectablemoieties of a multiplex assay using Linear Beacons. For example, twoLinear Beacons might be used to detect each of two different targetsequences wherein a fluorescein (green) labeled probe would be used todetect the first of the two target sequences and a rhodamine or Cy3(red) labeled probe would be used to detect the second of the two targetsequences. Consequently, a green, a red or a green and red signal in theassay would signify the presence of the first, second and first andsecond target sequences, respectively.

Arrays of Linear Beacons:

Arrays are surfaces to which two or more probes of interest have beenimmobilized at predetermined locations. Arrays comprising both nucleicacid and PNA probes have been described in the literature. The probesequences immobilized to the array are judiciously chosen to interrogatea sample which may contain one or more target sequences of interest.Because the location and sequence of each probe is known, arrays aregenerally used to simultaneously detect, identify or quantitate thepresence or amount of one or more target sequences in the sample. Thus,PNA arrays may be useful in diagnostic applications or in screeningcompounds for leads which might exhibit therapeutic utility.

For example, in a diagnostic assay a target sequence is captured by thecomplementary probe on the array surface and then the probe/targetsequence complex is detected using a secondary detection system. In oneembodiment the probe/target sequence complex is detected using a secondprobe which hybridizes to another sequence of the target molecule ofinterest. In another embodiment, a labeled antibody is used to detect,identify or quantitate the presence of the probe/target sequencecomplex.

Since the composition of the Linear Beacon is known at the location onthe surface of the array (because the PNA was synthesized or attached tothis position in the array), the composition of target sequence(s) canbe directly detected, identified or quantitated by determining thelocation of detectable signal generated in the array. Becausehybridization of the Linear Beacon to a target sequence isself-indicating, no secondary detection system is needed to analyze thearray for hybridization between the Linear Beacon and the targetsequence.

Arrays comprised of PNAs have the additional advantage that PNAs arehighly stable and should not be degraded by enzymes which degradenucleic acid. Therefore, PNA arrays should be reusable provided thenucleic acid from one sample can be striped from the array prior tointroduction of the second sample. Upon stripping of hybridized targetsequences, signal on the array of Linear Beacons should again becomereduced to background. Because PNAs are not degraded by heat orendonuclease and exonuclease activity, arrays of Linear Beacon should besuitable for simple and rapid regeneration by treatment with heat,nucleases or chemical denaturants such as aqueous solutions containingformamide, urea and/or sodium hydroxide.

Methods:

In yet another embodiment, this invention is directed to a method forthe detection, identification or quantitation of a target sequence in asample. The method comprises contacting the sample with a Linear Beaconand then detecting, identifying or quantitating the change in detectablesignal associated with at least one moiety of a Beacon Set wherebycorrelation between detectable signal and hybridization is possiblesince Linear Beacons are self-indicating. Because Linear Beacons areself-indicating, this method is particularly well suited to analysisperformed in a closed tube assay (a.k.a. “homogeneous assays”). Byclosed tube assay we mean that once the components of the assay havebeen combined, there is no need to open the tube or remove contents ofthe assay to determine the result. Since the tube need not, andpreferably will not, be opened to determine the result, there must besome detectable or measurable change which occurs and which can beobserved or quantitated without opening the tube or removing thecontents of the assay. Thus, most closed tube assays rely on a change influorescence which can be observed with the eye or otherwise detectedand/or quantitated with a fluorescence instrument which uses the tube asthe sample holder. Examples of such instruments include the Light Cyclerfrom Idaho Technologies and the Prism 7700 from Perkin Elmer.

Preferred closed tube assays of this invention comprise the detection ofnucleic acid target sequences which have been synthesized or amplifiedby operation of the assay. Non-limiting examples of preferred nucleicacid synthesis or nucleic acid amplification reactions are PolymeraseChain Reaction (PCR), Ligase Chain Reaction (LCR), Strand DisplacementAmplification (SDA), Transcription-Mediated Amplification (TMA), RollingCircle Amplification (RCA) and Q-beta replicase. The Linear Beaconspresent in the closed tube assay will generate detectable signal inresponse to target sequence production from the nucleic acid synthesisor nucleic acid amplification reaction occurring in the closed tubeassay. In a most preferred embodiment, the assay is an asymmetric PCRreaction (see: Example 19 of this specification).

Because the Linear Beacons of this invention can be designed to bestable to the enzymes found in the cell, this method is particularlywell suited to detecting a target sequence in a cell, tissue ororganism, whether living or not. Thus, in preferred embodiments, in-situhybridization is used as the assay format for detecting identifying orquantitating target organisms (see: Example 20 of this specification).Most preferably, fluorescence in-situ hybridization (FISH or PNA-FISH)is the assay format. Exemplary methods for performing PNA-FISH can befound in: Thisted et al. Cell Vision, 3:358-363 (1996) or WIPO PatentApplication WO97/18325, herein incorporated by reference.

Organisms which have been treated with the Linear Beacons of thisinvention can be detected by several exemplary methods. The cells can befixed on slides and then visualized with a microscope or laser scanningdevice. Alternatively, the cells can be fixed and then analyzed in aflow cytometer (see for example: Lansdorp et al.; WIPO PatentApplication; WO97/14026). Slide scanners and flow cytometers areparticularly useful for rapidly quantitating the number of targetorganisms present in a sample of interest.

Because the method of this invention may be used in a probe-basedhybridization assay, this invention will find utility in improvingassays used to detect, identify of quantitate the presence or amount ofan organism or virus in a sample through the detection of targetsequences associated with the organism or virus (see: U.S. Pat. No.5,641,631, entitled “Method for detecting, identifying and quantitatingorganisms and viruses” herein incorporated by reference). Similarly,this invention will also find utility in an assay used in the detection,identification or quantitation of one or more species of an organism ina sample (see: U.S. Pat. No. 5,288,611, entitled “Method for detecting,identifying and quantitating organisms and viruses” herein incorporatedby reference). This invention will also find utility in an assay used todetermine the effect of antimicrobial agents on the growth of one ormore microorganisms in a sample (see: U.S. Pat. No. 5,612,183, entitled“Method for determining the effect of antimicrobial agents on growthusing ribosomal nucleic acid subunit subsequence specific probes” hereinincorporated by reference). This invention will also find utility in anassay used to determine the presence or amount of a taxonomic group oforganisms in a sample (see: U.S. Pat. No. 5,601,984, entitled “Methodfor detecting the presence of amount of a taxonomic group of organismsusing specific r-RNA subsequences as probes” herein incorporated byreference).

When performing the method of this invention, it may be preferable touse one or more unlabeled or independently detectable probes in theassay to thereby suppress the binding of the Linear Beacon to anon-target sequence. The presence of the “blocking probe(s)” helps toincrease the discrimination of the assay and thereby improve reliabilityand sensitivity (signal to noise ratio).

In certain embodiments of this invention, one target sequence isimmobilized to a surface by proper treatment of the sample.Immobilization of the nucleic acid can be easily accomplished byapplying the sample to a membrane and then UV-crosslinking. For example,the samples may be arranged in an array so that the array can besequentially interrogated with one or more Linear Beacons to therebydetermine whether each sample contains one or more target sequence ofinterest.

In still another embodiment, the Linear Beacon is immobilized to asupport and the samples are sequentially interrogated to therebydetermine whether each sample contains a target sequence of interest. Inpreferred embodiments, the Linear Beacons are immobilized on an arraywhich is contacted with the sample of interest. Consequently, the samplecan be simultaneously analyzed for the presence and quantity of numeroustarget sequences of interest wherein the composition of the LinearBeacons are judiciously chosen and arranged at predetermined locationson the surface so that the presence, absence or amount of particulartarget sequences can be unambiguously determined. Arrays of LinearBeacons are particularly useful because no second detection system isrequired. Consequently, this invention is also directed to an arraycomprising two or more support bound Linear Beacons suitable fordetecting, identifying or quantitating a target sequence of interest.

Kits:

In yet another embodiment, this invention is directed to kits suitablefor performing an assay which detects the presence, absence or amount ofone or more target sequence which may be present in a sample. Thecharacteristics of Linear Beacons suitable for the detection,identification or quantitation of amount of one or more target sequencehave been previously described herein. Furthermore, methods suitable forusing the Linear Beacon components of a kit to detect, identify orquantitate one or more target sequence which may be present in a samplehave also been previously described herein.

The kits of this invention comprise one or more Linear Beacons and otherreagents or compositions which are selected to perform an assay orotherwise simplify the performance of an assay. Preferred kits containsets of Linear Beacons, wherein each of at least two Linear Beacons ofthe set are used to distinctly detect and distinguish between the two ormore different target sequences which may be present in the sample.Thus, the Linear Beacons of the set are preferably labeled withindependently detectable moieties so that each of the two or moredifferent target sequences can be individually detected, identified orquantitated (a multiplex assay).

Exemplary Applications for Using the Invention:

Whether support bound or in solution, the methods, kits and compositionsof this invention are particularly useful for the rapid, sensitive,reliable and versatile detection of target sequences which areparticular to organisms which might be found in food, beverages, water,pharmaceutical products, personal care products, dairy products orenvironmental samples. The analysis of preferred beverages include soda,bottled water, fruit juice, beer, wine or liquor products. Consequently,the methods, kits and compositions of this invention will beparticularly useful for the analysis of raw materials, equipment,products or processes used to manufacture or store food, beverages,water, pharmaceutical products, personal care products, dairy productsor environmental samples.

Whether support bound or in solution, the methods, kits and compositionsof this invention are particularly useful for the rapid, sensitive,reliable and versatile detection of target sequences which areparticular to organisms which might be found in clinical environments.Consequently, the methods, kits and compositions of this invention willbe particularly useful for the analysis of clinical specimens orequipment, fixtures or products used to treat humans or animals. Forexample, the assay may be used to detect a target sequence which isspecific for a genetically based disease or is specific for apredisposition to a genetically based disease. Non-limiting examples ofdiseases include, β-Thalassemia, sickle cell anemia, Factor-V Leiden,cystic fibrosis and cancer related targets such as p53, p10, BRC-1 andBRC-2.

In still another embodiment, the target sequence may be related to achromosomal DNA, wherein the detection, identification or quantitationof the target sequence can be used in relation to forensic techniquessuch as prenatal screening, paternity testing, identity confirmation orcrime investigation.

EXAMPLES

This invention is now illustrated by the following examples which arenot intended to be limiting in any way.

Example 1 Synthesis of N-α-(Fmoc)-N-ε-(NH₂)-L-Lysine-OH

To 20 mmol of N-α-(Fmoc)-N-ε-(t-boc)-L-lysine-OH was added 60 mL of 2/1dichloromethane (DCM)/trifluoroacetic acid (TFA). The solution wasallowed to stir until the tert-butyloxycarbonyl (t-boc) group hadcompletely been removed from the N-α-(Fmoc)-N-ε-(t-boc)-L-lysine-OH. Thesolution was then evaporated to dryness and the residue redissolved in15 mL of DCM. An attempt was then made to precipitate the product bydropwise addition of the solution to 350 mL of ethyl ether. Because theproduct oiled out, the ethyl ether was decanted and the oil put underhigh vacuum to yield a white foam. The white foam was dissolved in 250mL of water and the solution was neutralized to pH 4 by addition ofsaturated sodium phosphate (dibasic). A white solid formed and wascollected by vacuum filtration. The product was dried in a vacuum ovenat 35-40° C. overnight. Yield 17.6 mmol, 88%.

Example 2 Synthesis of N-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-OH

To 1 mmol of N-α-(Fmoc)-N-ε-(NH₂)-L-Lysine-OH (Example 1) was added 5 mLof N,N′-dimethylformamide (DMF) and 1.1 mmol of TFA. This solution wasallowed to stir until the amino acid had completely dissolved.

To 1.1 mmol of 4-((4-(dimethylamino)phenyl)azo)benzoic acid,succinimidyl ester (Dabcyl-NHS; Molecular Probes, P/N D-2245) was added4 mL of DMF and 5 mmol of diisopropylethylamine (DIEA). To this stirringsolution was added, dropwise, the N-α-(Fmoc)-N-ε-(NH₂)-L-Lysine-OHsolution prepared as described above. The reaction was allowed to stirovernight and was then worked up.

The solvent was vacuum evaporated and the residue partitioned in 50 mLof DCM and 50 mL of 10% aqueous citric acid. The layers were separatedand the organic layer washed with aqueous sodium bicarbonate and againwith 10% aqueous citric acid. The organic layer was then dried withsodium sulfate, filtered and evaporated to an orange foam. The foam wascrystallized from acetonitrile (ACN) and the crystals collected byvacuum filtration. Yield 0.52 mmol, 52%.

Example 3 Synthesis of N-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-PAL-Peg/PSSynthesis Support

The N-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-OH (Example 2) was used to preparea synthesis support useful for the preparation of C-terminal dabcylatedPNAs. The fluorenylmethoxycarbonyl (Fmoc) group of 0.824 g ofcommercially available Fmoc-PAL-Peg-PS synthesis support (PerSeptiveBiosystems, Inc.; P/N GEN913384) was removed by treatment, in a flowthrough vessel, with 20% piperidine in DCM for 30 minutes. The supportwas then washed with DCM. Finally, the support was washed with DMF anddried with a flushing stream of argon.

A solution containing 0.302 g N-α-(Fmoc)-N-ε-(dabcyl)-L-Lysine-OH, 3.25mL of DMF, 0.173 g [O-(7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), 0.101 mL DIEA and 0.068 mL 2,6-lutidine wasprepared by sequential combination of the reagents. This solution wasthen added to the washed synthesis support and allowed to react for 2hours. The solution was then flushed through the vessel with a stream ofargon and the support washed sequentially with DMF, DCM and DMF. Theresin was then dried with a stream of argon.

The support was the treated with 5 mL of standard commercially availablePNA capping reagent (PerSeptive Biosystems, Inc., P/N GEN063102). Thecapping reagent was then flushed from the vessel and the support waswashed with DMF and DCM. The support was then dried with a stream ofargon. Finally, the synthesis support was dried under high vacuum.

Final loading of the support was determined by analysis of Fmoc loadingof three samples of approximately 6-8 mg. Analysis determined theloading to be approximately 0.145 mmol/g.

This synthesis support was packed into an empty PNA synthesis column, asneeded, and used to prepare PNA oligomers having a C-terminal dabcylquenching moiety attached to the PNA oligomer through the 1-amino groupof the C-terminal L-lysine amino acid.

Example 4 Synthesis of PNA

PNAs were synthesized using commercially available reagents andinstrumentation obtained from PerSeptive Biosystems, Inc. Doublecouplings were often performed to insure that the crude product was ofacceptable purity. PNAs possessing a C-terminal dabcyl moiety wereprepared by performing the synthesis using the dabcyl-lysine modifiedsynthesis support prepared as described in Example 3 or by labeling theN-ε-amino group of the C-terminal lysine residue while the PNA was stillsupport bound as described in Example 10. All PNAs possessing both anN-terminal fluorescein moiety, as well as, a C-terminal dabcyl moietywere treated with the appropriate labeling reagents and linkers (asrequired) prior to cleavage from the synthesis support. PNAs comprisingan N-terminal Cy3 label (Amersham) were cleaved from the synthesissupport and HPLC purified prior to Cy3 labeling (see: Example 12).

Example 5 Preferred Method for Removal of the Fmoc Protecting Group

The synthesis support was treated with a solution of 25% piperidine inDMF for 5-15 minutes at room temperature. After treatment, the synthesissupport was washed and dried under high vacuum. The support was thentreated with the appropriate labeling reagent and/or cleaved from thesynthesis support.

Example 6 Synthesis of Fluorescein-O-Linker

To 7.5 mmol of N-(tert-butyloxycarbonyl)-8-amino-3,6-dioxaoctanoic acidstirring in 10 mL of DCM was added 50 mmol of TFA. The solution wasstirred at room temperature until the t-boc group was completelyremoved. The solvent was then removed by vacuum evaporation and theproduct was then resuspended in 10 mL of DCM. To this stirring solutionwas added, dropwise, a solution containing 7.5 mmol ofDi-O-pivaloyl-5(6)-carboxyfluorescein-NHS ester, 30 mmol ofN-methylmorpholine (NMM) and 20 mL of DCM. The reaction was allowed torun overnight and was then transferred to a separatory funnel in themorning.

This organic solution was washed with aqueous 10% citric acid two timesand then dried with sodium sulfate, filtered and evaporated to a brownfoam. The product was column purified using silica gel. A DCM mobilephase and stepwise methanol gradient was used to elute the product fromthe stationary phase. Yield 2.8 g of foam which was precipitated bydissolution in a minimal amount of DCM and dropwise addition of thatsolution to hexane. Yield 2.32 g white powder. The purity of the productwas not suitable for labeling so an additional reversed phasechromatographic separation was performed on a sample of this material.

One gram of the precipitated product was dissolved in 30 mL of a 50 mMaqueous triethylammonium acetate (pH 7) containing 40% acetonitrile.This solution was then added to a pre-equilibrated 2 g Waters Sep-PackVac 12 cc tC18 cartridge (P/N WAT043380) in 10, 3 mL aliquots. After theaddition of all loading solvent, two 3 mL aliquots of 50 mM aqueoustriethylammonium acetate (pH 7) containing 40% acetonitrile was loadedas a first wash. Two 3 mL aliquots of 50 mM aqueous triethylammoniumacetate (pH 7) containing 60% acetonitrile was then loaded as a secondwash. Finally, a single 3 mL aliquot of acetonitrile was used to elutematerial remaining on the column. The eluent of each aliquot wascollected individually and analyzed by HPLC for purity. The aliquotswere vacuum evaporated and the mass of each determined. Fractions ofsuitable purity were redissolved in DCM, the fractions were combined andprecipitated in hexane. Yield 0.232 g.

Example 7 General Procedure for N-terminal Labeling of Support Bound PNAwith Fluorescein-O-Linker

For N-terminal fluorescein labeling, the amino terminalfluorenylmethoxycarbonyl (Fmoc) group of several of the fully assembledPNA oligomers was removed by piperidine treatment and the resin waswashed and dried under vacuum. The resin was then treated for 20-30minutes with approximately 300 μL of a solution containing 0.07 MFluorescein-O-Linker, 0.06 M (HATU), 0.067 M DIEA and 0.1 M2,6-lutidine. After treatment the resin was washed and dried under highvacuum. The PNA oligomer was then cleaved, deprotected and purified asdescribed below.

Example 8 General Procedure for Labeling of Support Bound PNA with5(6)carboxyfluorescein-NHS

This method was used as an alternative to the procedure described inExample 7, for labeling PNAs with 5(6)-carboxyfluorescein. Thisprocedure requires that the N-terminus of the PNA oligomer be reactedwith Fmoc-8-amino-3,6-dioxaoctanoic acid prior to performing thelabeling reaction so that equivalent PNA constructs are prepared. Theamino terminal fluorenylmethoxycarbonyl (Fmoc) group of the fullyassembled PNA oligomer was removed by piperidine treatment and thesynthesis support was washed and dried under vacuum. The synthesissupport was then treated for 4-5 hours at 37° C. with approximately 300μL of a solution containing 0.1M 5(6)carboxyfluorescein-NHS (MolecularProbes, P/N C-1311), 0.3M DIEA and 0.3M 2,6-lutidine. After treatmentthe synthesis support was washed and dried under high vacuum. The PNAoligomer was then cleaved, deprotected and purified as described below.

More preferably, the synthesis support was then treated for 2-5 hours at30-37° C. with approximately 250 μL of a solution containing 0.08M5(6)carboxyfluorescein-NHS, 0.24M DIEA and 0.24M 2,6-lutidine.

Example 9 General Procedure for Labeling of Support Bound PNA with5(6)carboxyfluorescein

After proper reaction with linkers and removal of the terminal amineprotecting group, the resin was treated with 250 μL of a solutioncontaining 0.5M 5(6)carboxyfluorescein, 0.5MN,N′-diisopropylcarbodiimide, 0.5M 1-hydroxy-7-azabenzotriazole (HOAt)in DMF (see: Weber et al., Bioorganic & Medicinal Chemistry Letters, 8:597-600 (1998). After treatment the synthesis support was washed anddried under high vacuum. The PNA oligomer was then cleaved, deprotectedand purified as described below.

Note on Fluorescein Labeling: The fluorescein labeled PNAs describedherein were prepared using several different procedures. The differentprocedures have evolved to optimize fluorescein labeling conditions. Atthis time we prefer to use the procedure of Weber et al. for mostfluorescein labeling operations.

Example 10 General Procedure for Dabcyl Labeling of the N-ε-amino Groupof Support Bound L-Lysine

This procedure was used as an alternative to using the prederivatizedsupport when preparing dabcylated PNAs. This procedure has the advantagethat the lysine moiety (and therefore the attached dabcyl moiety) may beplaced at any location in the polymer including within the probingnucleobase sequence.

The resin (still in the synthesis column) was treated with 10 mL of asolution containing 1% trifluoroacetic acid, 5% triisopropylsilane (TIS)in dichloromethane by passing the solution through the column over aperiod of approximately 15 minutes. After treatment, the synthesissupport was washed with DMF. Prior to treatment with labeling reagentthe support was neutralized by treatment with approximately 10 mL of asolution containing 5% diisopropylethylamine in DMF. After treatment,the support was treated with Dabcyl-NHS (as a substitute for5(6)carboxyfluorescein-NHS in the procedure) essentially as described inExample 8.

Note: This procedure was only performed on PNA prepared usingFmoc-PAL-PEG/PS (PerSeptive P/N GEN913384). It was not performed withthe more acid labile Fmoc-XAL-PEG/PS (PerSeptive P/N GEN913394).

Example 11 General Procedure for Cleavage, Deprotection and Purification

The synthesis support (Fmoc-PAL-PEG/PS; P/N GEN913384) was removed fromthe synthesis cartridge, transferred to a Ultrafree spin cartridge(Millipore Corp., P/N SE3P230J3) and treated with a solution ofTFA/m-cresol (either of 7/3 or 8/2 (preferred)) for 1-3 hours. Thesolution was spun through the support bed and again the support wastreated with a solution of TFA/m-cresol for 1-3 hours. The solution wasagain spun through the support bed. The combined eluents (TFA/m-cresol)were then precipitated by addition of approximately 1 mL of diethylether. The precipitate was pelletized by centrifugation. The pellet wasthen resuspended in ethyl ether and pelletized two additional times. Thedried pellet was then resuspended in 20% aqueous acetonitrile (ACN)containing 0.1% TFA (additional ACN was added as necessary to dissolvethe pellet). The product was analyzed and purified using reversed phasechromatographic methods.

Note: Several PNAs were prepared using new product Fmoc-XAL-PEG/PSsynthesis support (P/N GEN 913394) available from PerSeptive Biosystems,Inc. This support has the advantage that the PNA can be removed morerapidly and under more mildly acid conditions. For PNAs prepared withFmoc-XAL-PEG/PS the support was treated as described above except that asolution of TFA/m-cresol 9/1 was generally used for a period of 10-15minutes (2×).

Example 12 Cy3 Labeling of PNAs

The purified amine containing PNA was dissolved in 1/1 DMF/water at aconcentration of 0.05 OD/mL to prepare a stock PNA solution. From thestock, approximately 30 mmole of PNA was added to a tube. To this tubewas then added 125 μL 0.1 M HEPES (pH 8.5), and enough 1/1 DMF/water tobring the total volume to 250 μL. This solution was thoroughly mixed. Toa prepackaged tube of Cy3 dye (Amersham), was added the entire 250 μLsolution prepared as described above. The tube was well mixed and thenallowed to react for 1 hour at ambient temperature.

After reaction, the solvent was removed by evaporation in a speed-vac.The pellet was then dissolved in 400 μL of a solution containing 3:11%aqueous TFA/ACN. Optionally the solution was then transferred to a 5000MW Ultrafree (Millipore, P/N UFC3LCC25) or a 3000 MW (Amicon, P/N 42404)filter to removed excess dye. The recovered product was then repurifiedusing reversed phase chromatographic methods.

Experiment 13 Analysis and Purification of PNA Oligomers

All PNA probes were analyzed and purified by reversed phase HPLC. Probecomposition was confirmed by comparison with theoretical calculatedmasses.

HPLC Procedures:

Generally, two different high performance liquid chromatography (HPLC)gradients were used to analyze and purify the PNA oligomers (Gradients A& B). Preparative purifications were scaled based on the analyticalanalysis conditions described in Gradients A & B. Gradient B wasdeveloped because initial purification using standard gradients(Gradient A) proved to be less than satisfactory. The experimentalconditions are as described below except that some attempts were made toimprove purifications by the addition of 20% formamide to the runningbuffers during some of the purifications. This procedure was abandonedsince it did not appear to produce any beneficial results. Curiouslyhowever, careful review of the data suggested that the HPLC artifactspreviously thought to correlate with the structure of certain probes(see: Provisional Patent Application No. 60/063,283 filed on Oct. 27,1997) was also found to correlate with the presence of formamide duringthe purification. Therefore, no correlation is now believed to existbetween structure of the PNA probe and the HPLC profiles observed forthe purified oligomers.

Gradients A & B

-   Buffer A=0.1% TFA in water.-   Buffer B=0.1% TFA in acetonitrile.-   Flow Rate: 0.2 mL/min.-   Column Temperature: 60° C.-   Instrument: Waters 2690 Alliance: Control by Waters Millennium    Software-   Stationary Phase: Waters Delta Pak C18, 300 Å, 5 μm, 2×150 mm (P/N    WAT023650)    Detection at 260 nm

Gradient Profile A Time (min.) Percent Buffer Percent Buffer B Curve0.00 100 0 0 4.00 100 0 6 22.00 80 20 6 38.00 40 60 6 40.00 20 80 11

Gradient Profile B Time (min) Percent Buffer A Percent Buffer B Curve0.00 90 10 0 40.00 60 40 6 50.00 20 80 6Mass Analysis:

Samples were analyzed using a linear Voyager Delayed Extraction MatrixAssisted Laser Desorption Ionization-Time Of Flight (DE MALDI-TOF) Massspectrometer (PerSeptive Biosystems, Inc.). Sinipinic acid was used asthe sample matrix and also used as one point for calibration of the massaxis. Bovine insulin was used as an internal calibration standard forthe second calibration point of the mass axis.

Samples were generally prepared for analysis by first preparing asolution of sinipinic acid at a concentration of 10 mg/mL in a 1:2mixture of acetonitrile and 0.1% aqueous trifluoroacetic acid. Next, aninsulin solution was prepared by dissolving 1 mg of bovine insulin(Sigma) in 0.1% aqueous trifluoroacetic acid. Finally, an insulin/matrixsolution was then prepared by mixing 9 parts of the sinipinic acidsolution to 1 part of the bovine insulin solution. Samples were preparedfor analysis by spotting 1 μL of the insulin/matrix solution followed byspotting 1 μL of diluted sample (approximately 0.1 to 1 OD per mL) ontothe mass spectrometer target. The instrument target was allowed to drybefore being inserted into the mass spectrometer.

Tables of PNA Oligomers Prepared for Study

TABLE 1A  Probes Prepared To Evaluate PNA Hairpins Probe SEQ ID Desc.CODE¹ PNA Probe Sequence NO: N-terminal Arm Forming Segments .001 5205Flu-O-TGG-AGO-OAC-GCC-ACC-AGC-TCC-AK(dabcyl)-NH₂ 13 .007 5105Flu-O-TGG-AGO-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH₂ 14 .010 5005Flu-O-TGG-AGA-CGC-CAC-CAG-CTC-CAK(dabcyl)-NH₂ 15 .002 3203Flu-O-TGG-OOA-CGC-CAC-CAG-CTC-CAK(dabcyl)-NH₂ 16 .008 3103Flu-O-TGG-OAC-GCC-ACC-AGC-TCC-AK(dabcyl)-NH₂ 17 .009 4004²Flu-O-TGG-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH₂ 18C-terminal Arm Forming Segments .018 7027Flu-O-ACG-CCA-CCA-GCT-CCA-OO-GTG-GCG-T-K(dabcyl)-NH₂ 19 .011A 5025Flu-O-ACG-CCA-CCA-GCT-CCA-OOG-GCG-TK(dabcyl)-NH₂ 20 .006 3023Flu-O-ACG-CCA-CCA-GCT-CCA-OOC-GTK(dabcyl)-NH₂ 21Probing Sequence External to the Arm Sequences .017 5115Flu-O-TAG-CAO-ACG-CCA-CCA-GCT-CCA-OTG-CTA-K(dabcyl)-NH₂ 22 .005 3113Flu-O-TAG-O-ACG-CCA-CCA-GCT-CCA-O-CTA-K(dabcyl)-NH₂ 23Control Probes; No Arm Forming Segments .003 0000Flu-O-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH₂ 24 .004 0110Flu-OO-ACG-CCA-CCA-GCT-CCA-OK(dabcyl)-NH₂ 25

The CODE is a simple means to determine the length of the complementarynucleobases at the amine and carboxyl termini of the PNA polymer and thenumber and location of any 8-amino-3,6-dioxaoctanoic acid flexiblelinker units. The probing nucleobase sequence is the same for all probeslisted in the table. The first digit in the CODE represents the lengthof the N-terminal arm segment which is complementary to the C-terminalarm segment. The second digit in the CODE represents the number offlexible linker units which link the N-terminal arm to the probingnucleobase sequence. The third digit in the CODE represents the numberof flexible linker units which link the C-terminal arm to the probingnucleobase sequence. The fourth digit in the CODE represents the lengthof the C-terminal arm segment which is complementary to the N-terminalarm segment. Consequently, the CODE can be used to visually compare thegeneral structure of the different PNA oligomers listed in Table 1.

A coincidental, 4 bp. overlap between the nucleobases at the amine andcarboxyl termini are present in this construct instead of the directlycomparable 3 bp. overlap.

TABLE 1B  Linear Beacons Prepared To Examine Properties Probe Desc.PNA Probe Sequence SEQ ID NO: PNA003.11(mer)Flu-O-GCC-ACC-AGC-TC-K(dabcyl)-NH₂ 26 PNA003.13(mer)Flu-O-CGC-CAC-CAG-CTC-C-K(dabcyl)-NH₂ 27 PNA003.15(mer)Flu-O-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH₂ 28 PNA003.17(mer)Flu-O-TAC-GCC-ACC-AGC-TCC-AA-K(dabcyl)-NH₂ 29 PNA003.MUFlu-O-ACG-CCA-CAA-GCT-CCA-K(dabcyl)-NH₂ 30 Cy3PNA003.15(mer)Cy3-O-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH₂ 31 Cy3SCBL03-15Cy3-O-CCA-GCA-TCA-CCA-GAC-K(dabcyl)-NH₂ 32

TABLE 1C  Linear Beacons Prepared To Evaluate PNA-FISH Assays Probe Target Desc. Organism PNA Probe Sequence SEQ ID NO: Pse16S32 PseudomonasFlu-O-CTG-AAT-CCA-GGA-GCA-K(dabcyl)-NH₂ 33 Pse16S34 PseudomonasFlu-O-AAC-TTG-CTG-AAC-CAC-K(dabcyl)-NH₂ 34 Bac16S19 BacillusFlu-O-CTT-TGT-TCT-GTC-CAT-K(dabcyl)-NH₂ 35

For Tables 1A, 1B and 1C, all PNA sequences are written from the amineto the carboxyl terminus. Abbreviations are:Flu=5-(6)-carboxyfluorescein,dabcyl=4-((4-(dimethylamino)phenyl)azo)benzoic acid,O=8-amino-3,6-dioxaoctanoic acid; K=the amino acid L-Lysine and Cy3 isthe cyanine 3 dye available from Amersham.

Example 14 Synthesis of DNA Oligonucleotides for Study

For this study, biotin labeled DNA oligonucleotides suitable as nucleicacids comprising a target sequence which are complementary to the PNAprobing sequence of the k-ras PNA probes were either synthesized usingcommercially available reagents and instrumentation or obtained fromcommercial vendors. Additionally, DNA oligomers of equivalent nucleobaselength and labeling configuration as compared with several LinearBeacons were prepared using the dabcyl synthesis support available fromGlen Research (P/N 20-5911) and other commercially available DNAreagents and instrumentation. The 5(6)carboxyfluorescein labeling of allDNAs was obtained using Fluoredite phosphoramidite (PerSeptiveBiosystems, Inc., P/N GEN080110) All DNAs were purified by conventionalmethods. The sequences of the DNA oligonucleotides prepared areillustrated in Tables 2A and 2B. Methods and compositions for thesynthesis and purification of synthetic DNAs are well known to those ofordinary skill in the art.

TABLE 2A  DNA Targets SEQ ID Description Target DNA Sequence NO:wt k-ras2 Biotin-GTG-GTA-GTT-GGA-GCT-GGT- 1 GGC-GTA-GGC-AAG-A SCBL-DNAGGT-AGT-GTC-TGG-TGA-TGC-TGG-AGG- 2 CAA

TABLE 2B  DNAs of Equivalent Subunit Length To Linear Beacons SEQ IDDescription DNA Probe Sequence NO: DNA003-11(mer)Flu-G-CCA-CCA-GCT-C-dabcyl 3 DNA003-13(mer) Flu-CG-CCA-CCA-GCT-CC-dabcyl4 DNA003-15(mer) Flu-ACG-CCA-CCA-GCT-CCA-dabcyl 5 DNA003-17(mer)Flu-TA-CGC-CAC-CAG-CTC-CAA- 6 dabcylDetailed Structural Analysis of PNA Oligomers Prepared for PNA MolecularBeacon Study

Example 15 Analysis of Fluorescent Thermal Profiles

General Experimental Procedure:

Fluorescent measurements were taken using a RF-5000spectrofluorophotometer (Shimadzu) fitted with a water jacketed cellholder (P/N 206-15439, Shimadzu) using a 1.6 mL, 10 mm path length cuvet(Stama Cells, Inc.). Cuvet temperature was modulated using a circulatingwater bath (Neslab). The temperature of the cuvet contents was monitoreddirectly using a thermocouple probe (Barnant; model No. 600-0000) whichwas inserted below liquid level by passing the probe tip through the capon the cuvet (custom manufacture).

Stock solution of HPLC purified PNA oligomer was prepared by dissolvingthe PNA in 50% aqueous N,N′-dimethylformamide (DMF). From each PNA stockwas prepared a solution of PNA oligomer, each at a concentration of 10pmol in 1.6 mL of Hyb. Buffer (50 mM Tris. HCl pH 8.3 and 100 mM NaCl)by serial dilution of purified PNA stock with Hyb. Buffer.

Samples were exited at 493 nm and the fluorescence measured at 521 nm.Data points were collected at numerous temperatures as the cuvet washeated and then again measured as the cuvet was allowed to cool.Generally, the bath temperature was sequentially increased by 5° C. andthen allowed to equilibrate before each data point was recorded.Similarly, to generate the cooling profile, the bath temperature wassequentially decreased by 5° C. and then allowed to equilibrate beforeeach data point was recorded.

Data Discussion:

Nucleic acid Molecular Beacons which form a hairpin structure areexpected to exhibit an increase in fluorescent intensity when heatedwhich is consistent with the melting of the hairpin stem and thephysical transition of the probe stem from a helix to a random coil.Consistent with any nucleic acid melting event, the process is expectedto be reversible thereby resulting in a decrease in fluorescence uponcooling of the sample caused by the resulting reformation of the helicalstructure. The expected melting phenomenon is documented for nucleicacid Molecular Beacons described by Tyagi et al (see: Tyagi et al NatureBiotechnology, 14: 303-308 (1996) at FIG. 3).

The results of the fluorescent thermal melting analysis of the PNAoligomers are summarized in the data presented in Table 3 and presentedgraphically in FIGS. 1A, 1B1, 1B2, 1B3 and 1C. With reference to Table3, there are three different general Thermal Profiles observed for thedifferent constructs and under the conditions examined. These arerepresented in Table 3 as Types A, B and C.

Fluorescent Thermal Profile Type A is characterized by a an increase influorescence intensity upon heating (melting) and a correlating decreasein fluorescence intensity upon cooling (reannealing). These results aresimilar to those published for nucleic acid Molecular Beacons which forma loop and hairpin stem structure. Thus, a Type A Fluorescent ThermalProfile is consistent with the formation of a stable hairpin stem andloop structure. This phenomenon is, therefore, believed to be caused bythe melting and reannealing of a stem and loop structure in the PNAMolecular Beacon. However, applicants only claim that a Type AFluorescent Thermal Profile is indicative of fairly reversiblefluorescence quenching, as other structures may be responsible for theobserved phenomenon.

Representatives of Type A Fluorescent Thermal Profiles are illustratedin FIG. 1A. The data presented in the Figure is for the PNA oligomers0.001, 0.007 and 0.002. Data for both the melting (open character) andthe reannealing (solid character) is presented. The sigmoidaltransitions are consistent with a melting a reannealing of a duplex.

Fluorescent Thermal Profile Type B is characterized by an increase influorescence intensity upon heating (melting), but, no substantialcorrelating decrease in fluorescence intensity upon cooling of thesample. Thus, under the conditions examined, the interactions whichinitially cause the quenching of fluorescence do not appear to bereadily reversible. Consequently, the data suggests that a PNA oligomerexhibiting a Type B Fluorescent Thermal Profile, does not exhibit allthe features of a True Molecular Beacon. Nonetheless, as seen by thehybridization assay data, a Type B Fluorescent Thermal Profile does notprohibit the PNA oligomer from functioning as a PNA Beacon.

Representatives of Type B Fluorescent Thermal Profiles are illustratedin FIGS. 1B1, 1B2 and 1B3. The data is presented in three sets so thateach trace may be more clearly viewed. The data presented in the Figuresare for the PNA oligomers 0.010, 0.008, 0.009 (FIG. 1B1), 0.018, 0.011A,0.017, (FIG. 1B2), and 0.003 and 0.004, (FIG. 1B3). Data for both themelting (open character) and the reannealing (solid character) ispresented.

Fluorescent Thermal Profile Type C is characterized by a high initialfluorescent intensity which initially decreases with heating and againdecreases even further upon cooling of the sample. The high initialfluorescent intensity suggests that this construct does not exhibit theinitial fluorescence quenching observed with most of the other PNAconstructs examined. The constant decrease in fluorescent intensity uponcooling is not well understood. Nevertheless, as seen by thehybridization assay data, a Type C, Fluorescent Thermal Profile does notprohibit the PNA oligomer from functioning as a PNA Beacon.Representatives of Type C Thermal Profiles are illustrated in FIG. 1C.The data presented in the FIG. 1C is for the PNA oligomers 0.005 and0.006. Data for both the melting (open character) and the reannealing(solid character) is presented.

TABLE 3 Summary of Data Compiled In Experiments 15-16 FluorescentThermal Hybridization Profile Profile Probe No. CODE Observed ObservedN-terminal Arm Forming Segments .001 5205 A A .007 5105 A A .010 5005 BA .002 3203 A A .008 3103 B A .009 4004 B A C-terminal Arm FormingSegments .018 7027 B A, B .011A 5025 B A .006 3023 C C Probing SequenceExternal To Arm Segments .017 5115 B B .005 3113 C C Control Probes: NoArm Forming Segments .003 0000 B B .004 0110 B B

Example 16 Analysis of Hybridization Assay Data

General Experimental Procedures:

All hybridization assay data was collected using a Wallac 1420 VICTORequipped with a F485 CW-lamp filter and a F535 Emission filter. The NUNCMaxiSorp, breakapart microtitre plate was used as the reaction vessel.Each microtitre plate was prewashed with Hyb. Buffer at room temperaturefor 15 minutes before the reaction components were added. Total reactionvolume was 100 μL in Hyb. Buffer.

Stock solution of purified PNA probe was prepared by dissolving the PNAin 50% aqueous N,N′-dimethylformamide (DMF). From this PNA Stock wasprepared a solution of each PNA at a concentration of 25 pmole/1 μL byserial dilution of the PNA Stock with 50% aqueous DMF.

Stock solution of purified wt k-ras2 DNA was prepared by dissolving thepurified DNA in TE (10 mM Tris. HCl pH 8.0; 1.0 mM EDTA, SigmaChemical). From this DNA Stock was prepared a solution of wt k-ras2 DNAat a concentration of 100 pmol/99 μL by serial dilution of the DNA Stockwith Hyb. Buffer.

Each reaction sample used for analysis was prepared by combining 1 μL ofthe appropriate PNA oligomer (25 pmole/μL) with either of 99 μL of wtk-ras2 DNA stock or 99 μL of Hyb. Buffer (control) as needed to prepare100 μL of sample.

Samples were mixed and then fluorescence intensity monitored with timeusing the Wallac VICTOR instrument. Samples were run in triplicate toinsure reproducible results. Data was acquired for 20-25 minutes afterthe reactants were mixed and then the wells were sealed and the plateheated to 42-50° C. in an incubator for 30-40 minutes. After cooling toambient temperature, the wells were unsealed and then another 10 datapoints were collected over approximately five minutes.

Data Discussion:

Nucleic acid Molecular Beacons which form a hairpin stem and loopstructure are expected to exhibit an increase in fluorescent intensityupon hybridization of the probing sequence to complementary nucleicacid. The expected increase in fluorescent intensity is documented forDNA Molecular Beacons described by Tyagi et al. (see: Tyagi et al.Nature Biotechnology, 14: 303-308 (1996)).

The results of the hybridization analysis of the PNA oligomers aresummarized in Table 3 and presented graphically in FIGS. 2A1, 2A2, 2A3,2B and 2C. With reference to Table 3, there are three different generalHybridization Profiles observed for the different constructs examined.These are represented in Table 3 as Types A, B and C. In FIG. 8, thesignal to noise ratio (before and after heating) for all probes examinedare graphically illustrated with the absolute values also presentedbelow the Figure.

Hybridization Profile Type A is characterized by the increase influorescence intensity in samples containing complementary target DNA ascompared with samples containing only the PNA oligomer. After heating,the fluorescent intensity of samples containing target sequenceincreases but the background fluorescence of the control sample(s) doesnot significantly change. Because the PNAs possess a very low inherentfluorescence, the probes which exhibit a Type A, Hybridization Profilegenerally have the highest signal to noise ratios. Representatives ofType A Hybridization Profiles are illustrated in FIGS. 2A1, 2A2 and 2A3.The data is presented in two separate graphical illustrations to clarifythe presentation. The data presented in the Figures is for the PNAoligomers 0.001, 0.007, 0.010 (FIG. 2A1), 0.002, 0.008, 0.009 (FIG.2A2), and 0.011A, 017 and 0.018 (FIG. 2A3).

Hybridization Profile Type B is characterized by the very rapid increasein fluorescence intensity in samples containing complementary target DNAas compared with samples containing only the PNA oligomer. Thefluorescence intensity quickly reaches a plateau which does notsignificantly change (if at all) after heating. The backgroundfluorescence of the control sample(s) does not change significantly evenafter heating. This suggest that the hybridization event rapidly, andwith little resistance, reaches a binding equilibrium without anyrequirement for heating. Representatives of Type B HybridizationProfiles are illustrated in FIG. 2B. The data presented in FIG. 2B isfor the PNA oligomers 0.018, 0.003 and 0.004 though PNA oligomer 0.018does not exhibit all the characteristics of a Type B HybridizationProfile. Specifically, the signal for probe 0.018 does not appear toincrease after heating (Type B) but the hybridization kinetics appear tobe more like a Type A Hybridization Profile.

Control probes 0.003 and 0.004 (herein referred to as Linear Beacons)exhibit a Type B Hybridization Profile. Thus, the rapid hybridizationkinetics of the Type B Hybridization Profile is probably the result ofhaving no hairpin stem, or any other strong force, which can stabilizethe non fluorescent polymer form. Nonetheless, the dynamic range (signalto noise ratio) observed in the hybridization assay of these probes istypically quite high and suggests that forces other than the hydrogenbonding of complementary nucleobases of arm segments can stabilize theinteractions between the donor and acceptor moieties. Applicants haveobserved that label/label interactions can be quite strong and may be animportant factor in this surprising result.

Though the background (noise) is higher for the 0.003 and 0.004 probes,as compared with the 0.001, 0.002, 0.007, 0.009 and 0.010 probes, thefluorescence intensity after hybridization is higher than that observedin any probes yet examined. As a result of the higher background, PNAoligomers 0.003 and 0.004 have a very favorable signal to noise ratio.This S/N ratio is nearly as favorable as any (and better than some) ofthe other PNA oligomers examined whether or not they possess armsegments. The data demonstrates that it is not necessary to have armforming segments to create a probe which exhibits an initial lowfluorescence intensity and a corresponding increase in fluorescencesignal upon the binding of the probe to a target sequence.

Hybridization Profile C is characterized by a moderate increase influorescence intensity in samples containing target DNA as compared withsamples containing only the PNA oligomer. The fluorescence intensityquickly reaches a plateau which does not significantly change (if atall) after heating. The background fluorescence of the control sample(s)is relatively high but does not change significantly even after heating.Hybridization Profiles B and C differ primarily because the backgroundfluorescence in the control samples, containing no target nucleic acid,are dramatically higher in Hybridization Profile Type C. Thehybridization data obtained for samples containing complementary nucleicacid, suggests that the hybridization event rapidly, and with littleresistance, reaches equilibrium. However, the very high backgroundsignal suggests that the forces which should hold the donor and acceptormoieties in close proximity are not strong enough in these constructs toeffectively quench the fluorescent signal. As a consequence of themoderate increase in fluorescence upon binding to the target sequenceand the higher than usual intrinsic fluorescence a PNA Molecular Beacon,which exhibits a Type C Hybridization Profile, has a very low signal tonoise ratio. Representatives of Type C Hybridization Profiles areillustrated in FIG. 2C. The data presented in FIG. 2C is for the PNAoligomers 0.006 and 0.005, respectively.

Summary of the Data Presented in Examples 15-16

The most surprising result of all the experiments performed byapplicants is that all of the PNA oligomers examined, including thecontrol probes 0.003 and 0.004, which have no arm forming segments,exhibit a correlation between increased fluorescence intensity andbinding of the probe to target sequence. Thus, it is not critical todesign PNAs to possess arm forming segments to thereby achieveconstructs which possess a very low intrinsic fluorescence, but whichbecome highly fluorescent upon hybridization to target sequence. This isa very surprising result in light of the teachings related to nucleicacid Molecular Beacons. Therefore, the Linear Beacons of this inventioncan be used to detect, identify or quantitate target sequences withoutany requirement that excess probes be separated from the probe/targetsequence complex prior to detection.

Example 17 Correlation of Linear Beacon Length with Noise (BackgroundFluorescence)

For this Example both DNA and PNA probes were compared to determine whateffect variations in length would have on the noise (baseline orbackground fluorescence) of native probe. Comparisons were made withrespect to changes in ionic strength (and minor change in pH), changesin the nature of the donor/acceptor pair and the presence or absence ofmagnesium.

Materials and Methods:

PNA probes PNA003-11, PNA003-13, PNA003-15, PNA003-17 and Cy3PNA003-15(see: Table 1B) and DNA probes DNA003-11, DNA003-13, DNA003-15 andDNA003-17 (see: Table 2B) were prepared as described. The purifiedprobes were diluted in TE Buffer (10 mM Tris-HCl pH 8.3, 1 mM EDTA) to aconcentration of 25 pmole/μL and then diluted to 25 pmole/1.6 mL withone of either Buffer A, B or C. Samples of the probes were prepared intriplicate and each 1.6 mL sample was analyzed using a Shimadzu RF-5000spectrofluorophotometer and a cell having a 10 mm path length. Forfluorescein labeled oligomers, the excitation wavelength was set at 493nm and the data was recorded for emission at 520 nm. For Cy3 labeledoligomers, the excitation wavelength was set at 545 nm and the data wasrecorded for emission at 560 nm. All data collected is recorded inrelative light units (RLU).

The background of each probe was examined in each of Buffers A, B and C.The results of the triplicate analyses were averaged and the dataobtained is graphically illustrated in FIG. 3 with the absolute valuefor the average RLU presented at the top of each bar. With reference toFIG. 3, the data is grouped into Buffers A, B and C for each probeexamined. With the exception of the Cy3PNA003-15 probe, all PNAs werelabeled at the N-terminus with 5(6)-carboxyfluorescein and at theC-terminus with dabcyl. The Cy3PNA003-15 probe differed from PNA003-15in that Cy3 had been substituted for 5(6)-carboxyfluorescein. All DNAswere labeled at the 5′-terminus with 5(6)-carboxyfluorescein and at the3′-terminus with a dabcyl. Given the commercially available chemistries,attempts were made to insure that label types and label spacing of theDNA and PNA probes were as comparable as reasonably possible.

Buffer Compositions:

-   Buffer A: 10 mM Sodium Phosphate, pH 7.0, 5 mM MgCl₂.-   Buffer B: 10 mM Sodium Phosphate, pH 7.0.-   Buffer C: 50 mM Tris-Cl pH 8.3, 100 mM NaCl.    Results and Discussion:

With reference to FIG. 3, the data for the fluorescein/dabcyl labeledDNA probes of 11, 13, 15 and 17 subunits in length are presented on theleft. From a cursory review of data there is a clear correlation betweenlength of the DNA oligonucleotide and the amount of noise (background).Specifically and without regard to the nature of the buffer, the noiseincreased substantially with each increase of two subunits of the DNAoligomer. This observation compares well with the reports of Mayrand etal. (see: U.S. Pat. No. 5,691,146, col. 7, lns. 8-24), Mathies et al.(see: U.S. Pat. No. 5,707,804 at col. 7, lns. 21-25) and Nazarenko etal. (see: Nucl. Acids Res. 25: at p. 2516, col. 2, lns. 36-40).

Regarding specific buffer effects, for all DNA oligomers, the noiseobserved in Buffer A was substantially lower than observed when theprobe was in buffers B or C. Since Buffers A and B are of comparableionic strength, clearly the presence of magnesium in Buffer Asubstantially reduced the noise of all probes. Though Buffers B and Cwere substantially different in ionic strength and marginally differentin pH, only a small increase in noise was observed with the change fromBuffer B to Buffer C. This change is more likely the result of the pHincrease since fluorescein will have a higher quantum yield at thehigher pH. Consequently, very little of the increase in noise which wasobserved between Buffers B and C is likely due to the increase in ionicstrength.

In summary, magnesium content and oligomer length appear to have asubstantial affect on noise (background fluorescence) of DNA probeswhereas variations in the ionic strength of the probe environmentappears to exhibit a lesser influence on noise.

With reference to FIG. 3, the data for the labeled PNA probes of 11, 13,15 and 17 subunits in length are presented on the right. From a cursoryreview of data there much less of a difference between the noise(background) observed for the probes of different length. Moreover,unlike DNA, there is no clear correlation between probe length and noiseintensity.

Regarding specific buffer effects, most dramatic of all was theconsistency of background irrespective of the nature of the buffer.Though there were minor differences, the absolute difference in noise(background fluorescence) measured in each of the three buffers wasremarkably small as compared with the DNA probes. Consequently, thenoise of PNA probes was found to be fairly independent of the length ofthe probe, the presence or absence of magnesium and the ionic strengthof the buffer. Again, the small increase in noise between Buffers B andC is likely a pH effect.

The data for the PNA probe, Cy3PNA003-15 can be most effectivelycompared with the data for PNA probe, PNA003-15, since only the donorfluorophores (Fluorescein to Cy3) differ. Though intensity of noise(background) cannot be directly correlated since the emission andexcitation wavelengths used to examine the fluorescein and Cy3 dyes aresubstantially different, for this Linear Beacon there is almost norelative difference in noise in each of the three buffers examined. Mostnotably, there is no substantial difference between the data for BuffersB and C. This supports the argument that the increases in noise observedfor the fluorescein probes is most likely a pH effect since the quantumyield of Cy3 should be less affected by the small differences in pH.Furthermore, since optimal excitation and emission wavelength for boththe fluorescein and Cy3 fluorophores were used for examination, thecomparatively low backgrounds for the Linear Beacons under fluorophoreoptimized conditions indicates that substantial quenching offluorescence occurs for both probes without regard to the nature of thespectral properties of the donor fluorophore and acceptor quencher.Taken as a whole the data indicates that the noise of the PNA probes aresubstantially independent of probe length, ionic strength, presence orabsence of magnesium and the spectral properties of the Beacon Set.

In summary, for PNA probes, the noise is substantially independent ofthe presence or absence of magnesium, oligomer length and ionic strengthas compared with DNA probes having the most similar length and labelingconfiguration. Linear Beacons also possess the unusual property thatenergy transfer can occurs without regard to the nature of the spectralproperties of the Beacon Set thereby indicating that the energy transferlikely occurs primarily by contact and not through FRET. Nevertheless,this data demonstrates a clear distinction in the structure and functionbetween the PNA probes and the DNA probes examined.

Example 18 Correlation of Linear Beacon Length with Signal to Noise in aHybridization Assay

For this Example both DNA and PNA probes were compared to determine whateffect variations in length would have on the signal to noise ratio ofthe native probe wherein the signal to noise ratio is derived from thesignal generated in the presence of target sequence as compared with thenoise or background fluorescent of the probe in the absence of targetsequence. Comparisons were made with respect to changes in probe length,ionic strength, changes in the nature of the donor/acceptor pair and thepresence or absence of magnesium. On a practical level, this datadiffers from that presented in Example 17 since it compares relativeperformance of the probes in a hybridization assay. For brevity, onlythe data for the 11-mer and 15-mer DNAs and PNAs is presented.

Materials and Methods:

PNA probes PNA003-11, PNA003-15, Cy3PNA003-15 (see: Table 1B) and DNAprobes DNA003-11 and DNA003-15 (see: Table 2B) were prepared asdescribed. The purified probes were diluted in TE Buffer (10 mM Tris-HClpH 8.3, 1 mM EDTA) to a concentration of 25 pmole/μL and then this stockwas further diluted to 25 pmole/50 μL with one of either Buffer A, B orC. The composition of Buffers A, B and C are described in Example 17.Samples of 50 μL of each probe in the appropriate Buffer was placed ineach of six wells in a microtitre plate such that for each probe, threehybridization reactions and three negative control reactions (used tomeasure the noise or background fluorescence) were performed. For eachof the hybridization reactions, 50 μL of target DNA (wt k-ras, Table2A), which had been prepared by dilution of the target DNA in TE bufferto 100 pmole/μL and subsequent dilution of this stock to 25 pmole/μLwith each of Buffers A, B or C, was added to each reaction. For eachcontrol, 50 μL of one of Buffers A, B or C was added. As a consequenceof the time necessary to pipette and mix the contents of the wells, allreagents had been mixed for approximately 1 minute prior to the firstfluorescence reading. All hybridization reactions were performed atambient temperature.

Hybridization data was collected using a Wallac 1420 Victor multilabelcounter. The fluorescent intensity of each well was measured for 0.1second. For all samples, 40 measurements were taken over a period ofapproximately 30 minutes. Consequently, the time dependence of thesignal to noise ratios were derived from the data collected over the 30minute period. Signal to noise ratios derived from the average of thethree hybridization reactions, as compared with the control reactions,is presented for: 1) the DNA and PNA 11-mers in FIGS. 4A and 4B: 2). theDNA and PNA 15-mers in FIGS. 4C and 4D: and 3). the 15-mer PNA probeCy3PNA003-15 in FIG. 4E.

Results and Discussion:

With reference to FIG. 4A, signal to noise ratio for the 30 minutes ofdata collected for the DNA 11 mer in each of Buffers A, B and C ispresented. Since a signal to noise ratio of 1 indicates no signal, themost striking result is the absence of any signal when Buffer B is used.By comparison the addition of magnesium (Buffer A) or the increase inionic strength and pH (Buffer C) results in a substantial improvement insignal to noise. Furthermore, the rate of increase in signal to noiseover time is quite distinct and can be used to monitor hybridizationrate kinetics.

The signal to noise ratio obtained for the PNA 11-mer in all buffers isgraphically presented in FIG. 4B. By comparison to the DNA 11-mer, asignal to noise ratio of greater than one was obtained under allconditions examined. Moreover, there was less of a dynamic range in thesignal to noise ratio for each of the three buffers examined (the rangeof S/N for the DNA 11-mer at 30 minutes was about 1 to 14 whereas therange of S/N for the PNA 11-mer at 30 minutes was about 3 to 6).Consequently, the signal to noise ratio for the PNA 11-mer, as comparedwith the DNA 11-mer, appears to be fairly independent of ionic strengthof its environment though there is at least some increase attributableto the change between Buffers B and C. Moreover, the signal to noiseratio of the PNA 11-mer appears to be completely independent of thepresence or absence of magnesium since the data for Buffers A and B isessentially the same.

Additionally, there is very little increase in signal to noise ratio forthe PNA 11-mer over time. Consequently, the data suggests that thehybridization kinetics of the Linear Beacon, PNA003.11, are extremelyrapid in all Buffers examined and that the hybridization has nearlyreached equilibrium within the first few minutes of the reaction.

With reference to FIGS. 4C and 4D, data for the DNA and PNA 15-mers,respectively, is graphically illustrated. Generally, all the dataobtained for the 15-mers parallels that observed for the 11-mers.Specifically, the signal to noise ratio for the DNA 15-mer in Buffer Bis 1, thereby indicating that no hybridization is detected within thebounds of the experiment. In Buffers A and C, the DNA 15-mer yields asignal to noise ratio which increases with time such that the kineticsof hybridization can be determined by analysis the data. Additionally,the dynamic range of the signal to noise ratio for the DNA 15-mer inBuffer A and C is between 6 and 11 as compared to the PNA 15-mer whereinthe signal to noise ratio is about 7 to 13.

Taken as a whole the data demonstrates that the signal to noise ratio ofthe PNA 15 mer is fairly independent of the presence or absence ofmagnesium as the data is essentially the same for Buffers A and B.Furthermore, the signal to noise ratio for the PNA 15-mer, as comparedwith the DNA 15-mer, appears to be fairly independent of ionic strengthof its environment though there is at least some increase attributableto the change between Buffers B and C. Finally, the data suggests thatthe hybridization kinetics of the Linear Beacon, PNA003-15, areextremely rapid in all Buffers examined as the hybridization has nearlyreached equilibrium within the first few minutes of the reaction ascompared with the hybridization kinetics of the DNA 15-mer which aresubstantially slower. With reference to FIG. 4E, the signal to noisedata for the Cy3 labeled PNA 15-mer, Cy3PNA003-15, is presented. Thedata for this probe can be most effectively compared with the data forPNA probe, PNA003-15, since only the donor fluorophore (Fluorescein toCy3) has been altered. A cursory review of the data indicates thatagain, a positive signal to noise ratio is obtained in all threeBuffers. The dynamic range of the signal to noise ratio is about 6 to 11which compares well with the data for PNA003-15 thereby indicating thatthere is not a substantial dependence on the presence or absence ofmagnesium or a substantial dependence on ionic strength. Furthermore,the data clearly demonstrates that no substantial overlap between theemission of the donor moiety and the absorbance of the acceptor moietyis required in a Linear Beacon since the signal to noise ratio isessentially the same for both PNA003-15 and Cy3PNA003-15 despite thevery different spectral characteristics of the fluorescein or Cy3 donormoieties of the donor/acceptor pairs (Beacon Set).

Curiously however, for the PNA probe Cy3PNA003-15, Buffers A and Boutperform Buffer C. This result is substantially different than thatobserved with all the fluorescein labeled probes (DNA or PNA).Consequently, the data suggests that the nature of the labels or labelpair (Beacon Set) may be the most significant factor affecting thedynamic range of the signal to noise ratios observed for a single probein the different Buffers. This data tends to suggest that much of thedynamic range in signal to noise may be related to the nature of thelabels thereby further strengthening the argument that structure andfunction of Linear Beacons are fairly independent of the presence orabsence of magnesium and the ionic strength of the environment, ascompared with DNA probes, since most of the dynamic range observed indifferent Buffers is likely attributable to the nature of the labels andnot the structure or function of the Linear Beacon. By comparison, thebroad dynamic range of the signal to noise ratio and the lengthdependency of the noise (see: Example 17) observed for the DNA probesindicates that the composition of the environment can have a substantialeffect on the structure and function of a DNA probe.

Example 19 Detection of PCR Amplicons Using Linear Beacons

For this example, asymmetric PCR was evaluated for comparison withtraditional PCR because asymmetric PCR yields a significant excess ofsingle stranded nucleic acid. Since it is possible to choose which ofthe strands of the amplicon are preferentially amplified by judiciousadjustment of the ratio of 5′ and 3′ primers, it was possible to designthe assay so that the target sequence to which the Linear Beaconhybridizes was contained within the over produced single strandednucleic acid of the asymmetric PCR assay.

Consequently, a Linear Beacon was designed to hybridize to one of thestrands of a region of dsDNA sought to be amplified. The Linear Beaconwas added to the PCR cocktail before thermocycling. Though LinearBeacons may hybridize to the target sequence during thermocycling,significant inhibition of the amplification process was not observed.Consequently, the PCR amplification was successfully monitored using thedetectable fluorescent signal of the Linear Beacon which was generatedin response to the activity of the PCR reaction. The data presentedconclusively demonstrates the feasibility of using Linear Beacons forthe detection of amplified nucleic acid in a closed tube (homogeneous)assay whether traditional or asymmetric PCR was used.

Materials and Methods:

PCR reactions were performed in mini-eppendorf tubes in a Perkin-Elmer2400 thermocycler. The PCR protocol involved a 5 second warm up to 94°C. (1st cycle only), followed by denaturing at 94° C. for 5 seconds,annealing at 55° C. for 30 seconds, and extension at 74° C. for 30seconds. The denaturation-annealing-extension cycle was repeated for 45cycles. Samples of 10 μL were withdrawn from each PCR reaction at theend of the 30 second extension step at cycles 30, 35, 40 and 45. All 10μL samples were placed in a 96 well conical bottom microtiter plate andfluorescence was monitored using a Wallac 1420 Victor™ Multilabel platereader. The average fluorescence intensity was recorded in relativelight units (RLU) over 1.0 second (excitation filter wavelength 485 nm;emission filter wavelength 535 nm).

All PCR reactions were derived from a single “master mix” to which wereadded either plasmid (for positive reactions) or plasmid buffer(negative reactions). PCR reactions containing 1 μL of plasmid DNA orplasmid buffer (10 mM TRIS-HCl pH 8.0, 1 mM EDTA) as a control, 50 pmoleof the 5′ primer, variable amounts the 3′ primer as described below, 1μL of 10 pmole/μL Linear Beacon, PNA003.MU (Table 1B) in 50% aqueousN,N′-dimethylformamide, 3 mM MgCl₂, 250 μM ATP, 250 μM CTP, 250 μM GTP,250 μM TTP, 2.85 units AmpliTaq DNA polymerase, 50 mM KCl, and 10 mMTRIS-HCl pH 8.3. in a total volume of 50 μL were prepared. The ratio of5′ primer to 3′ primer was either 1:1 (50 pmole 3′ primer), 10:1 (5pmole 3′ primer), or 100:1 (0.5 pmole 3′ primer).

The plasmid, pKRASMU, was generated by cloning a PCR amplicon from humanDNA into the pCR2.1 plasmid (Invitrogen). The human DNA was preparedfrom a cell line, Calu-1, which contains a point mutation at base 129 ofthe K-ras gene. Clones were screened by restriction fragment analysisand sequenced. Large preparations of the plasmid were generated andquantitated using standard techniques. The amplified region flanks theK-ras mutation and was 111 by in length. PCR reactions which were notthermocycled were used as fluorescence controls.

Probes and Primers and Targets:

5′ primer 5′ ATGACTGAATATAAACTTGT 3′ SEQ ID NO: 7 3′ primer 5′CTCTATTGTTGGATCATATT 3′ SEQ ID NO: 8dsDNA Template (amplified region only)

     <−3′ primer hyb. site −> 5′GAGATAACAACCTAGTATAAGCAGGTGTTTTACTAAGACTTA... SEQ ID NO: 9 3′CTCTATTGTTGGATCATATTCGTCCACAAAATGATTCTGAATT... SEQ ID NO: 10                             <−Linear Beacon Hyb. site−>   ...ATCGACTTAGCAGTTCCGTGAGAACGGATGCGGTGTTCGAGGT     ...AGCTGTATCGTCAAGGCACTCTTGCCTACGCCACAAGCTCCAAC   TGATGGTGTTCAAATATAAGTCAGTA 3′  SEQ ID NO: 11   TACCACAAGTTTATATTCAGTCAT   5′  SEQ ID NO: 12         <−5′primer hyb. site−>

As illustrated above, the Linear Beacon, PNA003.MU, was designed suchthat it does not overlap the primer regions.

Results and Discussion:

Table 4 presents the fluorescence data recorded for PCR reactions atcycles 30, 35, 40, and 45. For convenience of discussion, the rows ofthe Table have been assigned numbers 1-6 and the columns have beenassigned letters A-G. Average background, from controls which did notundergo thermocycling, has been subtracted from the data presented inthe Table.

TABLE 4 PCR Data A B C D E F G 1 Primers 5′:3′ 50:50 50:50 50:5 50:550:0.5 50:0.5 2 pKRASMU − + − + − + 3 30 Rnds 420 1458 166 8804 340 4504 35 Rnds 548 1746 440 11694 580 2458 5 40 Rnds 368 1308 −280 8552 122012 6 45 Rnds 386 1412 438 10698 636 3976

With reference to Table 4, the ratios of the 5′ and 3′ primers,respectively, which were used in each assay are listed in row 1. Thesymbol found on row 2 of the table is used to indicate the presence (+)or absence (−) of 1 fmole of the pKRASMU plasmid (PCR template) in eachassay. Rows 3-6 of column A indicate total cycles of PCR which wereperformed to generate the data presented.

Data for reactions containing no template (columns B, D and F) range invalue from −280 to 636, with an average of 338, whereas data forreactions containing template (columns C, E, and G) are significantlyhigher in all cases except row 3, column G. Additionally, the intensityof fluorescence of samples containing template exhibits a correlationbetween the ratio of primers and the number of PCR cycles. For example,the data for a standard PCR reaction (column C), where equivalentamounts of 5′ and 3′ primers are used, exhibited a fairly consistentfluorescence intensity at all cycles for which data was recorded. Bycomparison, the fluorescence for asymmetric PCR (column E), wherein the5′ to 3′ primer ratio was 10:1, was substantially more intense. Thisdata suggests that the 10:1 ratio of 5′ primer to 3′ primer facilitatesrobust amplification which significantly overexpresses the singlestranded nucleic acid containing the target sequence.

The fluorescence for asymmetric PCR (column 6), wherein the 5′ to 3′primer ratio was 100:1, was not as intense by comparison with the datain column G. However, the asymmetric PCR did exhibit a clear correlationbetween increasing number of PCR cycles and the intensity offluorescence. The lower fluorescence observed at a primer ratio of 100:1is likely the result of a lower total abundance of target sequencecontaining single stranded nucleic acid which is caused by having tenfold less 3′ primer in the initial cycles of the PCR amplificationreaction as compared with the sample whose data is presented in columnE.

FIG. 5 is a digital image of a photo of the sample (˜0 μL) remaining intubes 3 and 4 after 45 cycles of PCR. Tube 3 (left) corresponds to thedata presented in row 6, column D, and tube 4 (right) corresponds to thedata presented in row 6, column E. The photo was taken on a UVtransilluminator. Tube 4, which contained template, is fluorescent byvisual inspection whereas, tube 3, which was a control not containingtemplate is not visibly fluorescent thereby confirming a negative resultby mere visual inspection.

Taken as a whole, the data presented in this example demonstrates thatLinear Beacons can be used to detect nucleic acid which has beenamplified in a closed tube (homogeneous) assay. For asymmetric PCRreactions, the intensity of fluorescent signal was not onlysubstantially higher but asymmetric PCR was shown to exhibit acorrelation between the number of cycles performed and signal intensity.Thus, quantitation of amplified nucleic was possible using this method.

Example 20 PNA-FISH with Linear Beacons

Individual 3 mL cultures of bacteria were grown overnight in Tryptic SoyBroth (TSB) at 30° C. The OD₆₀₀ of each sample was measured and theneach culture was diluted into fresh TSB to an OD₆₀₀ of 0.5. Cultureswere allowed to double 3-4 times before harvesting. Cells from a 20 mLculture were pelleted by centrifugation at 8000 rpm. for 5 minutes,resuspended in 20 mL PBS (7 mM Na₂HPO₄; 3 mM NaH₂PO₄; 130 mM NaCl),pelleted again and resuspended in Fixation Buffer (3% paraformaldehydein PBS). The bacteria were incubated at room temperature for 30-60minutes before they were pelleted again (centrifugation at 8000 rpm for5 minutes) and after removal of the fixation solution, resuspended in 20mL of 50% aqueous ethanol. The fixed bacteria may either be used after30 minutes of incubation at ambient temperature or stored at −20° C. forseveral weeks prior to use.

For each assay, 100 μL of fixed cells in 50% aqueous ethanol wastransferred to a 1.5 mL microcentrifuge tube and centrifuged at 8000rpm. for 5 min. The aqueous ethanol was then removed and the pellet wasresuspended in 100 μL of sterile PBS and pelleted again bycentrifugation at 8000 rpm. for 5 min. The PBS was removed from thepellet, and the cells were resuspended in 100 μL of hybridization buffer(25 mM Tris-HCl, pH 9.0; 100 mM NaCl; 0.5% SDS) which contained theappropriate Linear Beacon(s) at a concentration of approximately 30pmol/mL. The hybridization reaction was performed at 50° C. for 30minutes. The Linear Beacons and their target organisms are listed inTable 1C. The Pseudomonas probes were used in a mixture of 1 to 1 foreach hybridization wherein the concentration of each probe was 30pmol/mL in each hybridization reaction. The sample was then centrifugedat 8000 R.P.M. for 5 min. The hybridization buffer was removed and thecells resuspended in 100 μL sterile TE-9.0 (10 mM Tris-HCl, pH 9.0; 1 mMEDTA).

An aliquot of 2 μL of this suspension of cells was placed on a glassslide, spread and allowed to dry. Lastly, 2 μL Vectashield (VectorLaboratories, P/NH-1000) was deposited over the dried cells and acoverslip was added and its position fixed using a couple of drops ofnail polish.

The slides were inspected using a Nikon fluorescent microscope equippedwith a 60× immersion oil objective, a 10× ocular (total enlargement is600 fold) and light filters obtained from Omega Optical (XF22 (green)and XF34 (red)). Electronic digital images of portions of the slideswere made using a SPOT CCD-camera and software obtained from DiagnosticInstruments, Inc., Sterling Heights, Mich. (USA).

The digital images obtained are presented in FIGS. 6A and 6B. Fixed E.coli, P. aeruginosa, and B. subtilis cells were all hybridized witheither a P. aeruginosa (FIG. 6A) or a B. subtilis (FIG. 6B) LinearBeacon as described above. In both FIGS. 6A and 6B, the red imagespresented in panels I, III and V are of cells stained with propidiumiodide which were visible using the red microscope filter. In both FIGS.6A and 6B, the green images presented in panels II, IV and VI are ofcells having a green fluorescence caused by hybridization of thefluorescein labeled Linear Beacon to the rRNA target sequence and whichare visible using the green microscope filter. For comparative purposes,the red and green images for each probe examined are of the same sectionof each slide and are presented one over the other in the Figures.

With reference to FIG. 6A, the cells of E. coli, P. aeruginosa and B.subtilis can be seen in the red images presented in panels I, III and V,respectively. The cells are red since the propidium iodide will stainall the bacterial which are present. With reference to Panels, II, IVand VI of FIG. 6A, green cells are most intensely visible only in panelIV thereby confirming that the Linear Beacon can be used to specificallyidentify the presence of the target organism P. aeruginosa.

With reference to FIG. 6B, again the cells of E. coli, P. aeruginosa andB. subtilis can be seen in the red images presented in panels I, III andV, respectively. With reference to Panels, II, IV and VI of FIG. 6B,green cells are most intensely visible only in panel VI therebyconfirming that the Linear Beacon can be used to specifically identifythe presence of the target organism B. subtilis.

In summary, the Linear Beacons directed to P. aeruginosa and Bacillusprovide for the unambiguous detection of target organisms even thoughthe protocol does not include any washing steps after the hybridizationreaction is performed.

Example 21 Correlation of Noise and Signal to Noise with NucleobaseSequence

This example was performed to determine whether the phenomena observedby applicants was sequence dependent. Therefore the nucleobase sequenceof PNA probe Cy3PNA003-15 (see: Table 1B) was rearranged to produce theprobe Cy3SCBL03-15 (see: Table 11B).

Materials and Methods:

The preparation, labeling and purification of PNA oligomers has beendescribed. The probe Cy3SCBL03-15 was examined in Buffers B and Cessentially as described in Example 18 of this specification using theDNA target SCBL-DNA (see: Table 2A). The data obtained is graphicallyrepresented in FIGS. 7A and 7B.

Results and Discussion:

With reference to FIG. 7A, the raw signal and noise data is illustratedfor the two buffer examined. As was observed for the probe Cy3PNA003-15,the results for probe Cy3SCBL03-15 appear to be substantiallyindependent of the buffer thereby confirming that ionic strength and aminimal pH change does not effect the results. With reference to FIG.7B, the signal to noise ratio is approximately 6-8 upon hybridization tothe target sequence. This correlates well the data presented in FIG. 4Efor the probe Cy3PNA003-15. Therefore, the data indicates that thephenomena observed by applicants is not substantially dependent upon thenucleobase sequence of the Linear Beacon.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the invention as defined by the appended claims. Those skilled in theart will be able to ascertain, using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed in the scope of the claims.

1. A method for determining a PCR amplified nucleic acid, said methodcomprising: a) contacting a sample comprising nucleic acid to be PCRamplified with at least one polymer comprising at least one linkedenergy donor moiety and at least one linked energy acceptor moietywherein said donor and acceptor moieties are separated by at least aportion of a probing nucleobase sequence and wherein said polymer is apeptide nucleic acid that does not form a stem and loop hairpin and isfurther characterized in that the efficiency of transfer of energybetween said donor and acceptor moieties, when the polymer is solvatedin aqueous solution, is substantially independent of at least twovariables selected from the group consisting of: i) nucleobase sequencelength separating the at least one energy donor moiety from the at leastone energy acceptor moiety; ii) spectral overlap of the at least onelinked energy donor moiety and the at least one linked energy acceptormoiety; iii) presence or absence of magnesium in the aqueous solution;and iv) the ionic strength of the aqueous solution; b) contacting thesample with primers and other reagents for PCR amplification; c) PCRamplifying the nucleic acid; d) determining hybridization of the polymerto a target sequence within the amplified nucleic acid wherein thetarget sequence present in the sample is correlated with a change indetectable signal associated with at least one donor or acceptor moietyof the polymer; and e) determining the presence, absence or amount ofPCR amplified nucleic acid in the sample, with the further proviso thatthe nucleic acid to be amplified is contained within a plasmid.
 2. Themethod of claim 1, wherein the PCR amplification is traditional.
 3. Themethod of claim 1, wherein the PCR amplification is asymmetric.
 4. Themethod of claim 1, wherein the PCR amplification is performed as aclosed tube (homogeneous) assay.
 5. The method of claim 1, wherein theamplified nucleic acid is quantitated.
 6. The method of claim 1, whereinthe presence or absence of the amplified nucleic acid is determined. 7.A method for determining a PCR amplified nucleic acid, said methodcomprising: a) contacting a sample comprising nucleic acid to be PCRamplified with a polymer, wherein said polymer is a petide nucleic acidcomprising: i) a probing nucleobase sequence for probing a targetsequence to which the probing nucleobase sequence is complementary orsubstantially complementary; ii) at least one energy donor moiety thatis linked to the probing nucleobase sequence; and iii) at least oneenergy acceptor moiety that is linked to the probing nucleobase sequencewherein the at least one donor moiety is separated from the at least oneacceptor moiety by at least a portion of the probing nucleobasesequence; b) contacting the sample with primers and other reagents forPCR amplification; c) PCR amplifying the nucleic acid; d) determininghybridization of the polymer to a target sequence within the amplifiednucleic acid wherein the target sequence present in the sample iscorrelated with a change in detectable signal associated with at leastone donor or acceptor moiety of the polymer; and e) determining thepresence, absence or amount of PCR amplified nucleic acid in the sample,with the further proviso that wherein the nucleic acid to be amplifiedis contained within a plasmid.
 8. The method of claim 7, wherein the PCRamplification is traditional.
 9. The method of claim 7, wherein the PCRamplification is asymmetric.
 10. The method of claim 7, wherein the PCRamplification is performed as a closed tube (homogeneous) assay.
 11. Themethod of claim 7, wherein the amplified nucleic acid is quantitated.12. The method of claim 7, wherein the presence or absence of theamplified nucleic acid is determined.